Formulations for neoplasia vaccines and methods of preparing thereof

ABSTRACT

The present invention relates to neoplasia vaccine or immunogenic composition formulation for the treatment or prevention of neoplasia in a subject and to methods of preparing thereof.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application claims priority and benefit of U.S. provisionalapplication Ser. No. 62/172,890 filed Jun. 9, 2015.

Reference is made to international patent application Serial No.PCT/US2014/068893 filed Dec. 5, 2014 and that claims priority to U.S.provisional patent application Ser. No. 61/913,172, filed Dec. 6, 2013.

FEDERAL FUNDING LEGEND

This invention was made with government support under grant numbersCA155010 and HL103532 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appln cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to formulations for the treatment ofneoplasia and methods of preparing thereof. More particularly, thepresent invention relates to the formulations for tumor vaccines fortreatment of neoplasia in a subject and methods of preparing thereof.

BACKGROUND OF THE INVENTION

Approximately 1.6 million Americans are diagnosed with neoplasia everyyear, and approximately 580,000 people in the United States are expectedto die of the disease in 2013. Over the past few decades there beensignificant improvements in the detection, diagnosis, and treatment ofneoplasia, which have significantly increased the survival rate for manytypes of neoplasia. However, only about 60% of people diagnosed withneoplasia are still alive 5 years after the onset of treatment, whichmakes neoplasia the second leading cause of death in the United States.

Currently, there are a number of different existing cancer therapies,including ablation techniques (e.g., surgical procedures, cryogenic/heattreatment, ultrasound, radiofrequency, and radiation) and chemicaltechniques (e.g., pharmaceutical agents, cytotoxic/chemotherapeuticagents, monoclonal antibodies, and various combinations thereof).Unfortunately, such therapies are frequently associated with seriousrisk, toxic side effects, and extremely high costs, as well as uncertainefficacy.

There is a growing interest in cancer therapies that seek to targetcancerous cells with a patient's own immune system (e.g., cancervaccines) because such therapies may mitigate/eliminate some of theherein-described disadvantages. Cancer vaccines are typically composedof tumor antigens and immunostimulatory molecules (e.g., cytokines orTLR ligands) that work together to induce antigen-specific cytotoxic Tcells that target and destroy tumor cells. Current cancer vaccinestypically contain shared tumor antigens, which are native proteins(i.e.—proteins encoded by the DNA of all the normal cells in theindividual) that are selectively expressed or over-expressed in tumorsfound in many individuals. While such shared tumor antigens are usefulin identifying particular types of tumors, they are not ideal asimmunogens for targeting a T-cell response to a particular tumor typebecause they are subject to the immune dampening effects ofself-tolerance. Vaccines containing tumor-specific and patient-specificneoantigens can overcome some of the disadvantages of vaccinescontaining shared tumor antigens.

In general, any vaccine should have a shelf-life long enough to ensurethat the vaccine will not degrade or deteriorate before use. Storagestability also requires that the components of the vaccine should notprecipitate from solution during storage. However, achieving adequatestorage stability can be difficult. Accordingly, new formulations forvaccines are needed.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

The present invention relates to neoplasia vaccines or immunogeniccompositions for the treatment of neoplasia, and more particularly tothe vaccine formulations comprising a pool of tumor-specific andpatient-specific neo-antigens for the treatment of tumors in a subject.

In one aspect, the invention provides a method of selecting a peptideinvolving: determining the isoelectric point (Pi) and hydrophobicity(HYDRO) of at least one peptide; and selecting the peptide when its Piand HYDRO is bounded by Pi ≥5 and HYDRO ≥−6.0, Pi ≥8 and HYDRO ≥−8.0, Pi≤5 and HYDRO ≥−5, or Pi ≥9 and HYDRO ≤−8.0, optionally when its Pi andHYDRO is bounded by Pi >7 and a HYDRO value of ≥−5.5. In someembodiments, the method involves determining the Pi and HYDRO of atleast two peptides, and selecting the peptide when its Pi and HYDRO isbounded by or closest to Pi ≥5 and HYDRO ≥−6.0, Pi ≥8 and HYDRO ≥−8.0,Pi ≤5 and HYDRO ≥−5, or Pi ≥9 and HYDRO ≤−8.0. In some relatedembodiments, the selected peptide is used in the methods describedherein (e.g., methods for preparing aqueous solutions, pharmaceuticalcompositions, immunogenic compositions, vaccine compositions, and thelike).

In one aspect, the invention provides a method of assessing thesolubility of a peptide in an aqueous solution involving: determiningthe isoelectric point (Pi) and hydrophobicity (HYDRO) of the peptide,wherein the peptide is soluble in the aqueous solution when its Pi andHYDRO is bounded by Pi ≥5 and HYDRO ≥−6.0, Pi ≥8 and HYDRO ≥−8.0, Pi ≤5and HYDRO ≥−5, or Pi ≥9 and HYDRO ≤−8.0, optionally when its Pi andHYDRO is bounded by Pi >7 and a HYDRO value of ≥−5.5.

In one aspect, the invention provides a method of preparing an aqueouspeptide solution involving: determining the isoelectric point (Pi) andhydrophobicity (HYDRO) of at least one peptide; selecting the peptidewhen its Pi and HYDRO is bounded by Pi ≥5 and HYDRO ≥−6.0, Pi ≥8 andHYDRO ≥−8.0, Pi ≤5 and HYDRO ≥−5, or Pi ≥9 and HYDRO ≤−8.0, optionallywhen its Pi and HYDRO is bounded by Pi >7 and a HYDRO value of ≥−5.5;and preparing an aqueous solution containing the peptide.

In one embodiment, the peptide or at least one peptide is aneo-antigenic peptide. In one embodiment, the peptide or at least onepeptide ranges from about 5 to about 50 amino acids in length. In oneembodiment, the peptide or at least one peptide ranges from about 15 toabout 35 amino acids in length. In one embodiment, the peptide or atleast one peptide is about 15 amino acids or less in length. In oneembodiment, the peptide or at least one peptide is between about 8 andabout 11 amino acids in length. In one embodiment, the peptide or atleast one peptide is 9 or 10 amino acids in length. In one embodiment,the peptide or at least one peptide is about 30 amino acids or less inlength. In one embodiment, the peptide or at least one peptide isbetween about 6 and about 25 amino acids in length. In one embodiment,the peptide or at least one peptide is between about 15 and about 24amino acids in length. In one embodiment, the peptide or at least onepeptide is between about 9 and about 15 amino acids in length.

In one embodiment, the aqueous solution contains a pH modifier. In oneembodiment, the pH modifier is a base. In one embodiment, the pHmodifier is a dicarboxylate or tricarboxylate salt. In one embodiment,the pH modifier is citrate. In another rembodiment, the pH modifier issuccinate. In one embodiment, the succinate contains sodium succinate.In one embodiment. In one embodiment, the succinate is present in theaqueous solution at a concentration from about 1 mM to about 10 mM. Inone embodiment, the succinate is present in the aqueous solution at aconcentration of about 2 mM to about 5 mM.

In one embodiment, the aqueous solution further contains dextrose,trehalose or sucrose. In one embodiment, the aqueous solution furthercontains dimethylsulfoxide.

In one embodiment, the aqueous solution further contains animmunomodulator or adjuvant.

In one embodiment, the aqueous solution is a pharmaceutical composition.In one embodiment, the aqueous solution is an immunogenic composition.In one embodiment, the aqueous solution is a vaccine composition.

In one embodiment, the aqueous solution is lyophilizable.

In one aspect, the invention provides a method of preparing an aqueousneo-antigenic peptide solution, the method involving: determining theisoelectric point (Pi) and hydrophobicity (HYDRO) of at least oneneo-antigenic peptide; selecting the at least one neo-antigenic peptideif its Pi and HYDRO is bounded by Pi ≥5 and HYDRO ≥−6.0, Pi ≥8 and HYDRO≥−8.0, Pi ≤5 and HYDRO ≥−5, or Pi ≥9 and HYDRO ≤−8.0, optionally whenits Pi and HYDRO is bounded by Pi >7 and a HYDRO value of ≥−5.5;preparing a solution containing the at least one neo-antigenic peptideor a pharmaceutically acceptable salt thereof; and combining thesolution containing the at least one neo-antigenic peptide or apharmaceutically acceptable salt thereof with a solution containingsuccinic acid or a pharmaceutically acceptable salt thereof, therebypreparing a peptide solution for a neoplasia vaccine. In one embodiment,the method further involves filtering the solution. In one embodiment,the method further involves lyophilizing the filtered neo-antigenicpeptide solution.

In one embodiment, the neo-antigenic peptide solution contains 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40neo-antigenic peptides each of which has been selected based on having aPi and a HYDRO bounded by Pi ≥5 and HYDRO ≥−6.0, Pi ≥8 and HYDRO ≥−8.0,Pi ≤5 and HYDRO ≥−5, or Pi ≥9 and HYDRO ≤−8.0, optionally when its Piand HYDRO is bounded by Pi >7 and a HYDRO value of ≥−5.5. In oneembodiment, the neo-antigenic peptide solution contains at least twoneo-antigenic peptides that have been selected based on having a Pi anda HYDRO bounded by Pi ≥5 and HYDRO ≥−6.0, Pi ≥8 and HYDRO ≥−8.0, Pi ≤5and HYDRO ≥−5, or Pi ≥9 and HYDRO ≤−8.0, optionally when its Pi andHYDRO is bounded by Pi >7 and a HYDRO value of ≥−5.5. In one embodiment,the neo-antigenic peptide solution of claim contains at least threeneo-antigenic peptides that have been selected based on having a Pi anda HYDRO bounded by Pi ≥5 and HYDRO ≥−6.0, Pi ≥8 and HYDRO ≥−8.0, Pi ≤5and HYDRO ≥−5, or Pi ≥9 and HYDRO ≤−8.0, optionally when its Pi andHYDRO is bounded by Pi >7 and a HYDRO value of ≥−5.5. In one embodiment,the neo-antigenic peptide solution contains at least four neo-antigenicpeptides that have been selected based on having a Pi and a HYDRObounded by Pi ≥5 and HYDRO ≥−6.0, Pi ≥8 and HYDRO ≥−8.0, Pi ≤5 and HYDRO≥−5, or Pi ≥9 and HYDRO ≤−8.0, optionally when its Pi and HYDRO isbounded by Pi >7 and a HYDRO value of ≥−5.5. In one embodiment, theneo-antigenic peptide solution contains at least five neo-antigenicpeptides that have been selected based on having a Pi and a HYDRObounded by Pi ≥5 and HYDRO ≥−6.0, Pi ≥8 and HYDRO ≥−8.0, Pi ≤5 and HYDRO≥−5, or Pi ≥9 and HYDRO ≤−8.0, optionally when its Pi and HYDRO isbounded by Pi >7 and a HYDRO value of ≥−5.5.

In one embodiment, the at least one neoantigenic peptide ranges fromabout 5 to about 50 amino acids in length. In one embodiment, the atleast one neoantigenic peptide ranges from about 15 to about 35 aminoacids in length. In one embodiment, the peptide or at least one peptideis about 15 amino acids or less in length. In one embodiment, thepeptide or at least one peptide is between about 8 and about 11 aminoacids in length. In one embodiment, the peptide or at least one peptideis 9 or 10 amino acids in length. In one embodiment, the peptide or atleast one peptide is about 30 amino acids or less in length. In oneembodiment, the peptide or at least one peptide is between about 6 andabout 25 amino acids in length. In one embodiment, the peptide or atleast one peptide is between about 15 and about 24 amino acids inlength. In one embodiment, the peptide or at least one peptide isbetween about 9 and about 15 amino acids in length.

In one embodiment, the neo-antigenic peptide solution contains a pHmodifier. In one embodiment, the pH modifier is a base. In oneembodiment, the pH modifier is a dicarboxylate or tricarboxylate salt.In one embodiment, the pH modifier is citrate. In one embodiment, the pHmodifier is succinate. In one embodiment, the succinate contains sodiumsuccinate. In one embodiment, the succinate is present in theformulation at a concentration from about 1 mM to about 10 mM. In oneembodiment, the succinate is present in the formulation at aconcentration of about 2 mM to about 5 mM.

In one embodiment, the neo-antigenic peptide solution further contains apharmaceutically acceptable carrier. In one embodiment, thepharmaceutically acceptable carrier contains dextrose. In oneembodiment, the pharmaceutically acceptable carrier contains trehalose.In one embodiment, the pharmaceutically acceptable carrier containssucrose. In one embodiment, the pharmaceutically acceptable carrierfurther contains dimethylsulfoxide. In one embodiment, the neo-antigenicpeptide solution is lyophilizable.

In one embodiment, the neo-antigenic peptide solution further containsan immunomodulator or adjuvant. In one embodiment, the immunodulator oradjuvant is selected from the group consisting of poly-ICLC, 1018 ISS,aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM,GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS,ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryllipid A, MontanideIMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51,OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®, vector system, PLGAmicroparticles, resiquimod, SRL172, Virosomes and other Virus-likeparticles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, and Aquila'sQS21 stimulon. In one embodiment, the immunomodulator or adjuvantcontains poly-ICLC.

In one embodiment, the neo-antigenic peptide solution contains: one tofive neo-antigenic peptides or pharmaceutically acceptable saltsthereof, wherein each neo-antigenic peptide have been selected based onhaving a Pi and a HYDRO bounded by Pi ≥5 and HYDRO ≥−6.0, Pi ≥8 andHYDRO ≥−8.0, Pi ≤5 and HYDRO ≥−5, or Pi ≥9 and HYDRO ≤−8.0, optionallywhen its Pi and HYDRO is bounded by Pi >7 and a HYDRO value of ≥−5.5;1-3% dimethylsulfoxide; 3.6-3.7% dextrose; 3.6-3.7 mM succinate acid ora salt thereof; 0.5 mg/ml poly I:poly C; 0.375 mg/ml poly-L-Lysine; 1.25mg/ml sodium carboxymethylcellulose; and 0.225% sodium chloride.

In one embodiment, neo-antigenic peptide solution contains each of theneo-antigenic peptides at a concentration of about 300 μg/ml.

In one embodiment, the neo-antigenic peptide solution is apharmaceutical composition. In one embodiment, the neo-antigenic peptidesolution is an immunogenic composition. In one embodiment, theneo-antigenic peptide solution is a vaccine composition.

In one aspect, the invention provides a method described hereincontaining administering a neo-antigenic peptide solution describedherein to a subject diagnosed as having a neoplasia, thereby treatingthe neoplasia.

In one aspect, the invention provides a neoplasia vaccine made by amethod described herein involving determining the isoelectric point (Pi)and hydrophobicity (HYDRO) of at least one peptide; and selecting thepeptide when its Pi and HYDRO is bounded by Pi ≥5 and HYDRO ≥−6.0, Pi ≥8and HYDRO ≥−8.0, Pi ≤5 and HYDRO ≥−5, or Pi ≥9 and HYDRO ≤−8.0,optionally when its Pi and HYDRO is bounded by Pi >7 and a HYDRO valueof ≥−5.5.

In one aspect, the invention provides a pharmaceutical compositioncomprising: at least one neo-antigenic peptide or a pharmaceuticallyacceptable salt thereof; a pH modifier; and a pharmaceuticallyacceptable carrier.

In certain embodiments the pharmaceutical composition includes at leastone neo-antigenic peptide or a pharmaceutically acceptable salt thereofthat is soluble. Soluble peptides may be identified experimentally.Soluble peptides may be identified based on the amino acid sequence ofeach peptide. In one embodiment, the pharmaceutical composition includesat least one neo-antigenic peptide or a pharmaceutically acceptable saltthereof with a specific isoelectric point (P_(i)). In one embodiment,the pharmaceutical composition includes at least one neo-antigenicpeptide or a pharmaceutically acceptable salt thereof with a specifichydrophobicity. Hydrophobicity may be expressed as a HYDRO value. TheHYDRO value may be determined by using known values of hydrophobicity orhydrophilicity of each amino acid side chain. The HYDRO value may bedetermined by identifying uninterrupted stretches of hydrophobic aminoacids in the peptide. The HYDRO value may be determined by adding thehydrophobicity of each amino acid in an uninterrupted stretch ofhydrophobic amino acids. The HYDRO value may be the sum of values in theuninterrupted stretch of hydrophobic amino acids with the highest degreeof hydrophobicity. In one embodiment, the peptide is soluble based upona combination of P_(i) and HYDRO value. The peptide may be bounded by Pi≥5 and HYDRO ≥−6.0, Pi ≥8 and HYDRO ≥−8.0, Pi ≤5 and HYDRO ≥−5, and Pi≥9 and HYDRO ≤−8.0. In preferred embodiments, the peptide is within anyof these range of values.

In certain embodiments, the pharmaceutical composition is a vaccinecomposition.

In certain embodiments, the pharmaceutical composition comprises atleast two neoantigenic peptides. In certain embodiments, thepharmaceutical composition comprises at least three neo-antigenicpeptides. In certain embodiments, the pharmaceutical compositioncomprises at least four neo-antigenic peptides. In certain embodiments,the pharmaceutical composition comprises at least five neo-antigenicpeptides. The neoplasia vaccine or immunogenic compositionadvantageously comprises at least four different neoantigens (and bydifferent antigens it is intended that each antigen has a differentneoepitope), e.g., at least 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24or 25 or 26 or 27 or 28 or 29 or 30 or 31 or 32 or 33 or 34 or 35 or 36or 37 or 38 or 39 or 40 or more different neoantigens can be in theneoplasia vaccine or immunogenic composition.

In certain embodiments, the neoantigenic peptide ranges from about 5 toabout 50 amino acids in length. In another related embodiment, theneoantigenic peptide ranges from about 15 to about 35 amino acids inlength. Typically, the length is greater than about 15 or 20 aminoacids, e.g., from 15 to 50 or about 75 amino acids.

In one embodiment, the neoplasia vaccine or immunogenic compositionfurther comprises a pH modifier and a pharmaceutically acceptablecarrier.

In certain embodiments, the pH modifier is a base. In certainembodiments, the pH modifier is a dicarboxylate or tricarboxylate salt.In certain embodiments, the pH modifier is succinate. In certainembodiments, the pH modifier is citrate.

In certain embodiments, the succinic acid or a pharmaceuticallyacceptable salt thereof comprises di sodium succinate.

In certain embodiments, succinate is present in the formulation at aconcentration from about 1 mM to about 10 mM. In certain embodiments,succinate is present in the formulation at a concentration of about 2 mMto about 5 mM.

In certain embodiments, the pharmaceutically acceptable carriercomprises water.

In certain embodiments, the pharmaceutically acceptable carrier furthercomprises dextrose.

In certain embodiments, the pharmaceutically acceptable carrier furthercomprises trehalose

In certain embodiments, the pharmaceutically acceptable carrier furthercomprises sucrose.

In certain embodiments, the pharmaceutically acceptable carrier furthercomprises dimethylsulfoxide.

In certain embodiments, the pharmaceutical composition further comprisesan immunomodulator or adjuvant. In one embodiment, the method furthercomprises administration of an immunomodulator or adjuvant. In anotherrelated embodiment, the immunomodulator or adjuvant is selected from thegroup consisting of poly-ICLC, 1018 ISS, aluminum salts, Amplivax, AS15,BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod,ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL,vector system, PLGA microparticles, resiquimod, SRL172, Virosomes andother Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan,Pam3Cys, and Aquila's QS21 stimulon. In another further embodiment, theimmunomodulator or adjuvant is poly-ICLC.

The dissolution of these polymers in water leads to an acid solutionwhich is neutralized, preferably to physiological pH, in order to givethe adjuvant solution into which the vaccine or immunogenic compositionor antigen(s) or vector(s) thereof is incorporated. The carboxyl groupsof the polymer are then partly in COO⁻.

Preferably, a solution of adjuvant according to the invention,especially of carbomer, is prepared in distilled water, preferably inthe presence of sodium chloride, the solution obtained being at acidicpH. This stock solution is diluted by adding it to the required quantity(for obtaining the desired final concentration), or a substantial partthereof, of water charged with salt such as NaCl, preferablyphysiological saline (NaCl 9 g/l), all at once or in several portionswith concomitant or subsequent neutralization (pH 7.3 to 7.4),preferably with a base such as NaOH. This solution at physiological pHis used as is to reconstitute the vaccine, especially stored infreeze-dried or lyophilized form.

The polymer concentration in the final vaccine composition is 0.01% to2% w/v, more particularly 0.06 to 1% w/v, preferably 0.1 to 0.6% w/v.

In another aspect, invention provides a pharmaceutical composition whichis a neoplasia vaccine, comprising: one to five neo-antigenic peptidesor pharmaceutically acceptable salts thereof; 1-3% dimethylsulfoxide;3.6-3.7% dextrose in water; 3.6-3.7 mM succinate acid or a salt thereof;0.5 mg/ml poly I:poly C; 0.375 mg/ml poly-L-Lysine; 1.25 mg/ml sodiumcarboxymethylcellulose; and 0.225% sodium chloride. In certainembodiments, each of the one to five neo-antigenic peptides orpharmaceutically acceptable salts thereof are each present at aconcentration of about 300 μg/ml.

In another aspect, the invention provides a method of preparing aneo-antigenic peptide solution for a neoplasia vaccine, the methodcomprising: providing a solution comprising at least one neo-antigenicpeptide or a pharmaceutically acceptable salt thereof; and combining thesolution comprising at least one neo-antigenic peptide or apharmaceutically acceptable salt thereof with a solution comprisingsuccinic acid or a pharmaceutically acceptable salt thereof, therebypreparing a peptide solution for a neoplasia vaccine.

In certain embodiments the method includes preparing at least oneneo-antigenic peptide or a pharmaceutically acceptable salt thereof thatis soluble. Soluble peptides may be determined experimentally. Peptidesmay be determined based on the amino acid sequence of each peptide. Inone embodiment, the pharmaceutical composition includes at least oneneo-antigenic peptide or a pharmaceutically acceptable salt thereof witha specific isoelectric point (P_(i)). In one embodiment, thepharmaceutical composition includes at least one neo-antigenic peptideor a pharmaceutically acceptable salt thereof with a specifichydrophobicity. Hydrophobicity may be expressed as a HYDRO value. TheHYDRO value may be determined by using known values of hydrophobicity orhydrophilicity of each amino acid side chain. The HYDRO value may bedetermined by identifying uninterrupted stretches of hydrophobic aminoacids in the peptide. The HYDRO value may be determined by adding thehydrophobicity of each amino acid in an uninterrupted stretch ofhydrophobic amino acids. The HYDRO value may be the sum of values in theuninterrupted stretch of hydrophobic amino acids with the highest degreeof hydrophobicity. The peptide may be bounded by Pi ≥5 and HYDRO ≥−6.0,Pi ≥8 and HYDRO ≥−8.0, Pi ≤5 and HYDRO ≥−5, and Pi ≥9 and HYDRO ≤−8.0.In preferred embodiments, the peptide is within any of these range ofvalues.

In certain embodiments, the solution comprising at least oneneo-antigenic peptide or a pharmaceutically acceptable salt thereofcomprises at least two (or 3, or 4, or 5) neo-antigenic peptides. Incertain embodiments, the peptide solution for a neoplasia vaccinecomprises water, dextrose or trehalose or sucrose, succinate, anddimethylsulfoxide. In certain embodiments, the method further comprises,after the step of combining, filtering the peptide solution for aneoplasia vaccine.

In another aspect, the invention provides a method of preparing aneoplasia vaccine, the method comprising: providing a peptide solutionfor a neoplasia vaccine; and combining the peptide solution with asolution of an immunodulator or adjuvant, thereby preparing a neoplasiavaccine.

In another aspect, the invention provides a neoplasia vaccine made byany method described herein (e.g., the method described above).

In another aspect, the invention provides a neo-antigenic peptidesolution for a neoplasia vaccine, comprising: at least one neo-antigenicpeptide or a pharmaceutically acceptable salt thereof; and succinic acidor a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a method of treating a subjectdiagnosed as having a neoplasia, the method comprising: administering apharmaceutical composition of the invention (e.g., a pharmaceuticalcomposition described herein) to the subject, thereby treating theneoplasia.

In certain embodiments, the method further comprises administering asecond pharmaceutical composition of the invention (e.g., apharmaceutical composition described herein) to the subject.

In certain embodiments, the method further comprises administering athird pharmaceutical composition of the invention (e.g., apharmaceutical composition described herein) to the subject.

In certain embodiments, the method further comprises administering afourth pharmaceutical composition of the invention (e.g., apharmaceutical composition described herein) to the subject.

The administering of the neoplasia vaccine or immunogenic compositioncan be on one time schedule, e.g., weekly, biweekly, every three weeks,monthly, bimonthly, every quarter year (every three months), every thirdof a year (every four months), every five months, twice yearly (everysix months), every seven months, every eight months, every nine months,every ten months, every eleven months, annually or the like.

The neoplasia vaccine or immunogenic composition can be administered viasubcompositions, each containing a portion of the neoantigens, andsub-compositions can be administered to different places on the subjector patient; for instance, a composition comprising 20 differentneoantigens, can be administered in four (4) subcompositions, eachcontaining 5 of the 20 different neoantigens, and the four (4)subcompositions can be administered so as to endeavor to deliver eachsubcomposition at or near a draining lymph node of the patient, e.g., toeach of the arms and legs (e.g., thigh or upper thigh or near buttocksor lower back on each side of the patient) so as to endeavor to delivereach subcomposition at or near a draining lymph node of the patient orsubject. Of course, the number of locations and hence number ofsubcompositions can vary, e.g., the skilled practitioner could consideradministration at or near the spleen to have a fifth point ofadministration, and the skilled practitioner can vary the locations suchthat only one, two or three are used (e.g., each arm and a leg, each oflegs and one arm, each of the legs and no arms, or only both arms).

The vaccine or immunogenic composition administered at theaforementioned various intervals can be different formulations, and thesubcompositions administered at different places on the subject orpatient during a single administration can be different compositions.For instance, a first administration can be of a whole antigen vaccineor immunogenic composition and a next or later administration can be ofa vector (e.g., viral vector or plasmid) that has expression ofantigen(s) in vivo. Likewise, in the administration of differentsubcompositions to different locations on the patient or subject, someof the subcompositions can comprise a whole antigen and some of thesubcompositions can comprise a vector (e.g., viral vector or plasmid)that has expression of antigen(s) in vivo. And some compositions andsubcompositions can comprise both vector(s) (e.g., viral vector orplasmid) that has/have expression of antigen(s) in vivo and wholeantigens. Some vectors (e.g., poxvirus) that have expression ofantigen(s) in vivo can have an immunostimulatory or adjuvanting effect,and hence compositions or subcompositions that contain such vectors canbe self-adjuvanting. Also, by changing up the nature of how the antigensare presented to the immune system, the administrations can “prime” andthen “boost” the immune system. And in this text, when there is mentionof a “vaccine” it is intended that the invention comprehends immunogeniccompositions, and when there is mention of a patient or subject it isintended that such an individual is a patient or subject in need of theherein disclosed treatments, administrations, compositions, andgenerally the subject invention.

Moreover, the invention applies to the use of any type of expressionvector, such as a viral expression vector, e.g., poxvirus (e.g.,orthopoxvirus or avipoxvirus such as vaccinia virus, including ModifiedVaccinia Ankara or MVA, MVA-BN, NYVAC according to WO-A-92/15672,fowlpox, e.g., TROVAX, canarypox, e.g., ALVAC (WO-A-95/27780 andWO-A-92/15672) pigeonpox, swinepox and the like), adenovirus, AAVherpesvirus, and lentivirus; or a plasmid or DNA or nucleic acidmolecule vector. Some vectors that are cytoplasmic, such as poxvirusvectors, may be advantageous. However adenovirus, AAV and lentivirus canalso be advantageous to use in the practice of the invention.

In a ready-for-use, especially reconstituted, vaccine or immunogeniccomposition, the vector, e.g., viral vector, is present in thequantities within the ambit of the skilled person from this disclosureand the knowledge in the art (such as in patent and scientificliterature cited herein).

Whole antigen or vector, e.g., recombinant live vaccines generally existin a freeze-dried form allowing their storage and are reconstitutedimmediately before use in a solvent or excipient, which can include anadjuvant as herein discussed.

The subject of the invention is therefore also a vaccination orimmunization set or kit comprising, packaged separately, freeze-driedvaccine and a solution, advantageously including an adjuvant compound asherein discussed for the reconstitution of the freeze-dried vaccine.

The subject of the invention is also a method of vaccination orimmunization comprising or consisting essentially of or consisting ofadministering, e.g., by the parenteral, preferably subcutaneous,intramuscular or intradermal, route or by the mucosal route a vaccine orimmunogenic composition in accordance with the invention at the rate ofone or more administrations. Optionally this method includes apreliminary step of reconstituting the freeze-dried vaccine orimmunogenic composition (e.g., if lyophilized whole antigen or vector)in a solution, advantageously also including an adjuvant.

In one embodiment, the subject is suffering from a neoplasia selectedfrom the group consisting of: Non-Hodgkin's Lymphoma (NHL), clear cellRenal Cell Carcinoma (ccRCC), melanoma, sarcoma, leukemia or a cancer ofthe bladder, colon, brain, breast, head and neck, endometrium, lung,ovary, pancreas or prostate. In another embodiment, the neoplasia ismetastatic. In a further embodiment, the subject has no detectableneoplasia but is at high risk for disease recurrence. In a furtherrelated embodiment, the subject has previously undergone autologoushematopoietic stem cell transplant (AHSCT).

In one embodiment, administration of the neoplasia vaccine orimmunogenic composition is in a prime/boost dosing regimen. In anotherembodiment, administration of the neoplasia vaccine or immunogeniccomposition is at weeks 1, 2, 3 or 4 as a prime. In another furtherembodiment, administration of the neoplasia vaccine or immunogeniccomposition is at months 2, 3, 4 or 5 as a boost.

In one embodiment, the vaccine or immunogenic composition isadministered at a dose of about 10 μg-1 mg per 70 kg individual as toeach neoantigenic peptide. In another embodiment, the vaccine orimmunogenic composition is administered at an average weekly dose levelof about 10 μg-2000 μg per 70 kg individual as to each neoantigenicpeptide.

In one embodiment, the vaccine or immunogenic composition isadministered intravenously or subcutaneously.

In another aspect, the invention provides a neo-antigenic peptidesolution for a neoplasia vaccine, comprising: at least one neo-antigenicpeptide or a pharmaceutically acceptable salt thereof; and succinic acidor a pharmaceutically acceptable salt thereof.

The invention comprehends performing methods as in U.S. patentapplication No. 20110293637, incorporated herein by reference, e.g., amethod of identifying a plurality of at least 4 subject-specificpeptides and preparing a subject-specific immunogenic composition thatupon administration presents the plurality of at least 4subject-specific peptides to the subject's immune system, wherein thesubject has a tumor and the subject-specific peptides are specific tothe subject and the subject's tumor, said method comprising:

(i) identifying, including through

-   -   nucleic acid sequencing of a sample of the subject's tumor and    -   nucleic acid sequencing of a non-tumor sample of the subject,        a plurality of at least 4 tumor-specific non-silent mutations        not present in the non-tumor sample; and

(ii) selecting from the identified non-silent mutations the plurality ofat least 4 subject-specific peptides, each having a different tumorneo-epitope that is an epitope specific to the tumor of the subject,from the identified plurality of tumor specific mutations,

wherein each neo-epitope is an expression product of a tumor-specificnon-silent mutation not present in the non-tumor sample, eachneo-epitope binds to a HLA protein of the subject, and selectingincludes

-   -   determining binding of the subject-specific peptides to the HLA        protein, and

(iii) formulating the subject-specific immunogenic composition foradministration to the subject so that upon administration the pluralityof at least 4 subject-specific peptides are presented to the subject'simmune system,

wherein the selecting or formulating comprises at least one of:

-   -   including in the subject-specific immunogenic composition a        subject-specific peptide that includes an expression product of        an identified neo-ORF, wherein a neo-ORF is a tumor-specific        non-silent mutation not present in the non-tumor sample that        creates a new open reading frame, and    -   including in the subject-specific immunogenic composition a        subject-specific peptide that includes an expression product of        an identified point mutation and has a determined binding to the        HLA protein of the subject with an IC50 less than 500 nM,        whereby, the plurality of at least 4 subject-specific peptides        are identified, and the subject-specific immunogenic composition        that upon administration presents the plurality of at least 4        subject-specific peptides to the subject's immune system,        wherein the subject-specific peptides are specific to the        subject and the subject's tumor, is prepared; or a method of        identifying a neoantigen comprising:        a. identifying a tumor specific mutation in an expressed gene of        a subject having cancer;        b. wherein when said mutation identified in step (a) is a point        mutation:

i. identifying a mutant peptide having the mutation identified in step(a), wherein said mutant peptide binds to a class I HLA protein with agreater affinity than a wild-type peptide; and has an IC50 less than 500nm;

c. wherein when said mutation identified in step (a) is a splice-site,frameshift, read-through or gene-fusion mutation:

i. identifying a mutant polypeptide encoded by the mutation identifiedin step (a), wherein said mutant polypeptide binds to a class I HLAprotein; or a method of inducing a tumor specific immune response in asubject comprising administering one or more peptides or polypeptidesidentified and an adjuvant; or a method of vaccinating or treating asubject for cancer comprising:

a. identifying a plurality of tumor specific mutations in an expressedgene of the subject wherein when said mutation identified is a.

i. point mutation further identifying a mutant peptide having the pointmutation; and/or

ii. splice-site, frameshift, read-through or gene-fusion mutationfurther identifying a mutant polypeptide encoded by the mutation;

b. selecting one or more mutant peptides or polypeptides identified instep (a) that binds to a class I HLA protein;c. selecting the one or more mutant peptides or polypeptides identifiedin step (b) that is capable of activating anti-tumor CD8 T-cells; andd. administering to the subject the one or more peptides orpolypeptides, autologous dendritic cells or antigen presenting cellspulsed with the one or more peptides or polypeptides selected in step(c); or preparing a pharmaceutical composition comprising one identifiedpeptide(s), and performing method(s) as herein discussed. Thus, theneoplasia vaccine or immunogenic composition herein can be as in U.S.patent application No. 20110293637.

Accordingly, it is an object of the invention to not encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) is provided by the Office upon request and payment ofthe necessary fee.

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings, incorporated herein by reference, wherein:

FIG. 1 shows a flow process for making a personalized cancer vaccine orimmunogenic composition.

FIG. 2 shows a flow process for pre-treatment steps for generating acancer vaccine or immunogenic composition for a cancer patient.

FIG. 3 illustrates an immunization schedule based on a prime booststrategy according to an exemplary embodiment of the present invention.Multiple immunizations may occur over the first −3 weeks to maintain anearly high antigen exposure during the priming phase of immune response.Patients may then be rested for eight weeks to allow memory T cells todevelop and these T cells will then be boosted in order to maintain astrong ongoing response.

FIG. 4 shows a time line indicating the primary immunological endpointaccording to an exemplary aspect of the invention.

FIG. 5 shows a schematic depicting drug product processing of individualneoantigenic peptides into pools of 4 subgroups according to anexemplary embodiment of the invention.

FIG. 6 shows the results of quantitative PCR to assess the levels ofinduction of a number of key immune markers after stimulation of mousedendritic cells using a neoantigenic formulation.

FIG. 7 shows MDSC analysis of 5% Dextrose and 0.8% DMSO.

FIG. 8 shows MDSC analysis of 10% Trehalose and 0.8% DMSO.

FIG. 9 shows MDSC analysis of 10% Sucrose and 0.8% DMSO.

FIG. 10 shows the pressure profile of an exemplary lyophilization.

FIG. 11 shows the temperature profile of an exemplary lyophilization.

FIG. 12 shows the physical appearance of lyophilized cake usingexemplary formulations of the invention.

FIG. 13 shows an example of how the HYDRO value is determined for agiven peptide with the amino acid sequenceKYNDFDSEPMFLFIVFSHGILVNHMLIVVM (SEQ ID NO:1).

FIG. 14 shows a chart plotting HYDRO versus P_(i) for a set of peptides.

FIG. 15 shows a chart plotting HYDRO versus P_(i) for a larger set ofpeptides including the peptides in FIG. 14.

DETAILED DESCRIPTION OF THE INVENTION Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined herein:

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of thestated value. Unless otherwise clear from context, all numerical valuesprovided herein are modified by the term about.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a,” “an,” and “the” areunderstood to be singular or plural.

By “agent” is meant any small molecule chemical compound, antibody,nucleic acid molecule, or polypeptide, or fragments thereof.

By “ameliorate” is meant decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a disease (e.g.,a neoplasia, tumor, etc.).

By “alteration” is meant a change (increase or decrease) in theexpression levels or activity of a gene or polypeptide as detected bystandard art known methods such as those described herein. As usedherein, an alteration includes a 10% change in expression levels,preferably a 25% change, more preferably a 40% change, and mostpreferably a 50% or greater change in expression levels.

By “analog” is meant a molecule that is not identical, but has analogousfunctional or structural features. For example, a tumor specificneo-antigen polypeptide analog retains the biological activity of acorresponding naturally-occurring tumor specific neo-antigenpolypeptide, while having certain biochemical modifications that enhancethe analog's function relative to a naturally-occurring polypeptide.Such biochemical modifications could increase the analog's proteaseresistance, membrane permeability, or half-life, without altering, forexample, ligand binding. An analog may include an unnatural amino acid.

The term “neoantigen” or “neoantigenic” means a class of tumor antigensthat arises from a tumor-specific mutation(s) which alters the aminoacid sequence of genome encoded proteins.

By “neoplasia” is meant any disease that is caused by or results ininappropriately high levels of cell division, inappropriately low levelsof apoptosis, or both. For example, cancer is an example of a neoplasia.Examples of cancers include, without limitation, leukemia (e.g., acuteleukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acutemyeloblastic leukemia, acute promyelocytic leukemia, acutemyelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, chronic leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin'sdisease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavychain disease, and solid tumors such as sarcomas and carcinomas (e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma,meningioma, melanoma, neuroblastoma, and retinoblastoma).Lymphoproliferative disorders are also considered to be proliferativediseases.

The term “neoplasia vaccine” is meant to refer to a pooled sample ofneoplasia/tumor specific neoantigens, for example at least two, at leastthree, at least four, at least five, or more neoantigenic peptides. A“vaccine” is to be understood as meaning a composition for generatingimmunity for the prophylaxis and/or treatment of diseases (e.g.,neoplasia/tumor). Accordingly, vaccines are medicaments which compriseantigens and are intended to be used in humans or animals for generatingspecific defense and protective substance by vaccination. A “neoplasiavaccine composition” can include a pharmaceutically acceptableexcipient, carrier or diluent.

The term “pharmaceutically acceptable” refers to approved or approvableby a regulatory agency of the Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, including humans.

A “pharmaceutically acceptable excipient, carrier or diluent” refers toan excipient, carrier or diluent that can be administered to a subject,together with an agent, and which does not destroy the pharmacologicalactivity thereof and is nontoxic when administered in doses sufficientto deliver a therapeutic amount of the agent.

A “pharmaceutically acceptable salt” of pooled tumor specificneoantigens as recited herein may be an acid or base salt that isgenerally considered in the art to be suitable for use in contact withthe tissues of human beings or animals without excessive toxicity,irritation, allergic response, or other problem or complication. Suchsalts include mineral and organic acid salts of basic residues such asamines, as well as alkali or organic salts of acidic residues such ascarboxylic acids. Specific pharmaceutical salts include, but are notlimited to, salts of acids such as hydrochloric, phosphoric,hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic,formic, toluenesulfonic, methanesulfonic, benzene sulfonic, ethanedisulfonic, 2-hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic,citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic,pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic,phenylacetic, alkanoic such as acetic, HOOC—(CH2)n-COOH where n is 0-4,and the like. Similarly, pharmaceutically acceptable cations include,but are not limited to sodium, potassium, calcium, aluminum, lithium andammonium. Those of ordinary skill in the art will recognize from thisdisclosure and the knowledge in the art that further pharmaceuticallyacceptable salts for the pooled tumor specific neoantigens providedherein, including those listed by Remington's Pharmaceutical Sciences,17th ed., Mack Publishing Company, Easton, Pa., p. 1418 (1985). Ingeneral, a pharmaceutically acceptable acid or base salt can besynthesized from a parent compound that contains a basic or acidicmoiety by any conventional chemical method. Briefly, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in anappropriate solvent.

By a “polypeptide” or “peptide” is meant a polypeptide that has beenseparated from components that naturally accompany it. Typically, thepolypeptide is isolated when it is at least 60%, by weight, free fromthe proteins and naturally-occurring organic molecules with which it isnaturally associated. Preferably, the preparation is at least 75%, morepreferably at least 90%, and most preferably at least 99%, by weight, apolypeptide. An isolated polypeptide may be obtained, for example, byextraction from a natural source, by expression of a recombinant nucleicacid encoding such a polypeptide; or by chemically synthesizing theprotein. Purity can be measured by any appropriate method, for example,column chromatography, polyacrylamide gel electrophoresis, or by HPLCanalysis.

As used herein, the terms “prevent,” “preventing,” “prevention,”“prophylactic treatment,” and the like, refer to reducing theprobability of developing a disease or condition in a subject, who doesnot have, but is at risk of or susceptible to developing a disease orcondition.

The term “prime/boost” or “prime/boost dosing regimen” is meant to referto the successive administrations of a vaccine or immunogenic orimmunological compositions. The priming administration (priming) is theadministration of a first vaccine or immunogenic or immunologicalcomposition type and may comprise one, two or more administrations. Theboost administration is the second administration of a vaccine orimmunogenic or immunological composition type and may comprise one, twoor more administrations, and, for instance, may comprise or consistessentially of annual administrations. In certain embodiments,administration of the neoplasia vaccine or immunogenic composition is ina prime/boost dosing regimen.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50,as well as all intervening decimal values between the aforementionedintegers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,and 1.9. With respect to sub-ranges, “nested sub-ranges” that extendfrom either end point of the range are specifically contemplated. Forexample, a nested sub-range of an exemplary range of 1 to 50 maycomprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

A “receptor” is to be understood as meaning a biological molecule or amolecule grouping capable of binding a ligand. A receptor may serve, totransmit information in a cell, a cell formation or an organism. Thereceptor comprises at least one receptor unit and frequently containstwo or more receptor units, where each receptor unit may consist of aprotein molecule, in particular a glycoprotein molecule. The receptorhas a structure that complements the structure of a ligand and maycomplex the ligand as a binding partner. Signaling information may betransmitted by conformational changes of the receptor following bindingwith the ligand on the surface of a cell. According to the invention, areceptor may refer to particular proteins of MHC classes I and IIcapable of forming a receptor/ligand complex with a ligand, inparticular a peptide or peptide fragment of suitable length.

A “receptor/ligand complex” is also to be understood as meaning a“receptor/peptide complex” or “receptor/peptide fragment complex,” inparticular a peptide- or peptide fragment-presenting MHC molecule ofclass I or of class II.

By “reduces” is meant a negative alteration of at least 10%, 25%, 50%,75%, or 100%.

By “reference” is meant a standard or control condition.

A “reference sequence” is a defined sequence used as a basis forsequence comparison. A reference sequence may be a subset of, or theentirety of, a specified sequence; for example, a segment of afull-length cDNA or genomic sequence, or the complete cDNA or genomicsequence. For polypeptides, the length of the reference polypeptidesequence will generally be at least about 10-2,000 amino acids,10-1,500, 10-1,000, 10-500, or 10-100. Preferably, the length of thereference polypeptide sequence may be at least about 10-50 amino acids,more preferably at least about 10-40 amino acids, and even morepreferably about 10-30 amino acids, about 10-20 amino acids, about 15-25amino acids, or about 20 amino acids. For nucleic acids, the length ofthe reference nucleic acid sequence will generally be at least about 50nucleotides, preferably at least about 60 nucleotides, more preferablyat least about 75 nucleotides, and even more preferably about 100nucleotides or about 300 nucleotides or any integer thereabout or therebetween.

By “specifically binds” is meant a compound or antibody that recognizesand binds a polypeptide, but which does not substantially recognize andbind other molecules in a sample, for example, a biological sample.

Nucleic acid molecules useful in the methods of the invention includeany nucleic acid molecule that encodes a polypeptide of the invention ora fragment thereof. Such nucleic acid molecules need not be 100%identical with an endogenous nucleic acid sequence, but will typicallyexhibit substantial identity. Polynucleotides having “substantialidentity” to an endogenous sequence are typically capable of hybridizingwith at least one strand of a double-stranded nucleic acid molecule. By“hybridize” is meant pair to form a double-stranded molecule betweencomplementary polynucleotide sequences (e.g., a gene described herein),or portions thereof, under various conditions of stringency. (See, e.g.,Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507).

For example, stringent salt concentration will ordinarily be less thanabout 750 mM NaCl and 75 mM trisodium citrate, preferably less thanabout 500 mM NaCl and 50 mM trisodium citrate, and more preferably lessthan about 250 mM NaCl and 25 mM trisodium citrate. Low stringencyhybridization can be obtained in the absence of organic solvent, e.g.,formamide, while high stringency hybridization can be obtained in thepresence of at least about 35% formamide, and more preferably at leastabout 50% formamide. Stringent temperature conditions will ordinarilyinclude temperatures of at least about 30° C., more preferably of atleast about 37° C., and most preferably of at least about 42° C. Varyingadditional parameters, such as hybridization time, the concentration ofdetergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion orexclusion of carrier DNA, are well known to those skilled in the art.Various levels of stringency are accomplished by combining these variousconditions as needed. In a preferred: embodiment, hybridization willoccur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. Ina more preferred embodiment, hybridization will occur at 37° C. in 500mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/mldenatured salmon sperm DNA (ssDNA). In a most preferred embodiment,hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodiumcitrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variationson these conditions will be readily apparent to those skilled in theart.

For most applications, washing steps that follow hybridization will alsovary in stringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude a temperature of at least about 25° C., more preferably of atleast about 42° C., and even more preferably of at least about 68° C. Ina preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a more preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art. Hybridization techniques are well known to those skilled inthe art and are described, for example, in Benton and Davis (Science196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology,Wiley Interscience, New York, 2001); Berger and Kimmel (Guide toMolecular Cloning Techniques, 1987, Academic Press, New York); andSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York.

The term “subject” refers to an animal which is the object of treatment,observation, or experiment. By way of example only, a subject includes,but is not limited to, a mammal, including, but not limited to, a humanor a non-human mammal, such as a non-human primate, bovine, equine,canine, ovine, or feline.

By “substantially identical” is meant a polypeptide or nucleic acidmolecule exhibiting at least 50% identity to a reference amino acidsequence (for example, any one of the amino acid sequences describedherein) or nucleic acid sequence (for example, any one of the nucleicacid sequences described herein). Preferably, such a sequence is atleast 60%, more preferably 80% or 85%, and more preferably 90%, 95% oreven 99% identical at the amino acid level or nucleic acid to thesequence used for comparison.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. In an exemplary approach to determining thedegree of identity, a BLAST program may be used, with a probabilityscore between e⁻³ and e⁻¹⁰⁰ indicating a closely related sequence.

A “T-cell epitope” is to be understood as meaning a peptide sequencethat can be bound by MHC molecules of class I or II in the form of apeptide-presenting MHC molecule or MHC complex and then, in this form,be recognized and bound by naïve T-cells, cytotoxic T-lymphocytes orT-helper cells.

The terms “treat,” “treated,” “treating,” “treatment,” and the like aremeant to refer to reducing or ameliorating a disorder and/or symptomsassociated therewith (e.g., a neoplasia or tumor). “Treating” includesthe concepts of “alleviating”, which refers to lessening the frequencyof occurrence or recurrence, or the severity, of any symptoms or otherill effects related to a cancer and/or the side effects associated withcancer therapy. The term “treating” also encompasses the concept of“managing” which refers to reducing the severity of a particular diseaseor disorder in a patient or delaying its recurrence, e.g., lengtheningthe period of remission in a patient who had suffered from the disease.It is appreciated that, although not precluded, treating a disorder orcondition does not require that the disorder, condition, or symptomsassociated therewith be completely eliminated.

The term “therapeutic effect” refers to some extent of relief of one ormore of the symptoms of a disorder (e.g., a neoplasia or tumor) or itsassociated pathology. “Therapeutically effective amount” as used hereinrefers to an amount of an agent which is effective, upon single ormultiple dose administration to the cell or subject, in prolonging thesurvivability of the patient with such a disorder, reducing one or moresigns or symptoms of the disorder, preventing or delaying, and the likebeyond that expected in the absence of such treatment. “Therapeuticallyeffective amount” is intended to qualify the amount required to achievea therapeutic effect. A physician or veterinarian having ordinary skillin the art can readily determine and prescribe the “therapeuticallyeffective amount” (e.g., ED50) of the pharmaceutical compositionrequired. For example, the physician or veterinarian could start dosesof the compounds of the invention employed in a pharmaceuticalcomposition at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved.

The pharmaceutical compositions typically should provide a dosage offrom about 0.0001 mg to about 200 mg of compound per kilogram of bodyweight per day. For example, dosages for systemic administration to ahuman patient can range from 0.01-10 μg/kg, 20-80 μg/kg, 5-50 μg/kg,75-150 μg/kg, 100-500 μg/kg, 250-750 μg/kg, 500-1000 μg/kg, 1-10 mg/kg,5-50 mg/kg, 25-75 mg/kg, 50-100 mg/kg, 100-250 mg/kg, 50-100 mg/kg,250-500 mg/kg, 500-750 mg/kg, 750-1000 mg/kg, 1000-1500 mg/kg, 1500-2000mg/kg, 5 mg/kg, 20 mg/kg, 50 mg/kg, 100 mg/kg, of 200 mg/kg.Pharmaceutical dosage unit forms are prepared to provide from about0.001 mg to about 5000 mg, for example from about 100 to about 2500 mgof the compound or a combination of essential ingredients per dosageunit form.

A “vaccine” is to be understood as meaning a composition for generatingimmunity for the prophylaxis and/or treatment of diseases (e.g.,neoplasia/tumor). Accordingly, vaccines are medicaments which compriseantigens and are intended to be used in humans or animals for generatingspecific defense and protective substance by vaccination.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

The present invention relates to vaccines and methods for the treatmentof neoplasia, and more particularly tumors, by administering atherapeutically effective amount of a pharmaceutical composition (e.g.,a cancer vaccine) comprising a plurality of neoplasia/tumor specificneo-antigens to a subject (e.g., a mammal such as a human). As describedin more detail herein, whole genome/exome sequencing may be used toidentify all, or nearly all, mutated neoantigens that are uniquelypresent in a neoplasia/tumor of an individual patient, and that thiscollection of mutated neoantigens may be analyzed to identify aspecific, optimized subset of neoantigens for use as a personalizedcancer vaccine or immunogenic composition for treatment of the patient'sneoplasia/tumor. For example, a population of neoplasia/tumor specificneoantigens may be identified by sequencing the neoplasia/tumor andnormal DNA of each patient to identify tumor-specific mutations, and thepatient's HLA allotype can be identified. The population ofneoplasia/tumor specific neoantigens and their cognate native antigensmay then be subject to bioinformatic analysis using validated algorithmsto predict which tumor-specific mutations create epitopes that couldbind to the patient's HLA allotype. Based on this analysis, a pluralityof peptides corresponding to a subset of these mutations may be designedand synthesized for each patient, and pooled together for use as acancer vaccine or immunogenic composition in immunizing the patient. Theneo-antigens peptides may be combined with an adjuvant (e.g., poly-ICLC)or another anti-neoplastic agent. Without being bound by theory, theseneo-antigens are expected to bypass central thymic tolerance (thusallowing stronger anti-tumor T cell response), while reducing thepotential for autoimmunity (e.g., by avoiding targeting of normalself-antigens).

The immune system can be classified into two functional subsystems: theinnate and the acquired immune system. The innate immune system is thefirst line of defense against infections, and most potential pathogensare rapidly neutralized by this system before they can cause, forexample, a noticeable infection. The acquired immune system reacts tomolecular structures, referred to as antigens, of the intrudingorganism. There are two types of acquired immune reactions, whichinclude the humoral immune reaction and the cell-mediated immunereaction. In the humoral immune reaction, antibodies secreted by B cellsinto bodily fluids bind to pathogen-derived antigens, leading to theelimination of the pathogen through a variety of mechanisms, e.g.complement-mediated lysis. In the cell-mediated immune reaction, T-cellscapable of destroying other cells are activated. For example, ifproteins associated with a disease are present in a cell, they arefragmented proteolytically to peptides within the cell. Specific cellproteins then attach themselves to the antigen or peptide formed in thismanner and transport them to the surface of the cell, where they arepresented to the molecular defense mechanisms, in particular T-cells, ofthe body. Cytotoxic T cells recognize these antigens and kill the cellsthat harbor the antigens.

The molecules that transport and present peptides on the cell surfaceare referred to as proteins of the major histocompatibility complex(MHC). MHC proteins are classified into two types, referred to as MHCclass I and MHC class II. The structures of the proteins of the two MHCclasses are very similar; however, they have very different functions.Proteins of MHC class I are present on the surface of almost all cellsof the body, including most tumor cells. MHC class I proteins are loadedwith antigens that usually originate from endogenous proteins or frompathogens present inside cells, and are then presented to naïve orcytotoxic T-lymphocytes (CTLs). MHC class II proteins are present ondendritic cells, B-lymphocytes, macrophages and other antigen-presentingcells. They mainly present peptides, which are processed from externalantigen sources, i.e. outside of the cells, to T-helper (Th) cells. Mostof the peptides bound by the MHC class I proteins originate fromcytoplasmic proteins produced in the healthy host cells of an organismitself, and do not normally stimulate an immune reaction. Accordingly,cytotoxic T-lymphocytes that recognize such self-peptide-presenting MHCmolecules of class I are deleted in the thymus (central tolerance) or,after their release from the thymus, are deleted or inactivated, i.e.tolerized (peripheral tolerance). MHC molecules are capable ofstimulating an immune reaction when they present peptides tonon-tolerized T-lymphocytes. Cytotoxic T-lymphocytes have both T-cellreceptors (TCR) and CD8 molecules on their surface. T-Cell receptors arecapable of recognizing and binding peptides complexed with the moleculesof MHC class I. Each cytotoxic T-lymphocyte expresses a unique T-cellreceptor which is capable of binding specific MHC/peptide complexes.

The peptide antigens attach themselves to the molecules of MHC class Iby competitive affinity binding within the endoplasmic reticulum, beforethey are presented on the cell surface. Here, the affinity of anindividual peptide antigen is directly linked to its amino acid sequenceand the presence of specific binding motifs in defined positions withinthe amino acid sequence. If the sequence of such a peptide is known, itis possible to manipulate the immune system against diseased cellsusing, for example, peptide vaccines.

One of the critical barriers to developing curative and tumor-specificimmunotherapy is the identification and selection of highly specific andrestricted tumor antigens to avoid autoimmunity. Tumor neoantigens,which arise as a result of genetic change (e.g., inversions,translocations, deletions, missense mutations, splice site mutations,etc.) within malignant cells, represent the most tumor-specific class ofantigens. Neoantigens have rarely been used in cancer vaccine orimmunogenic compositions due to technical difficulties in identifyingthem, selecting optimized neoantigens, and producing neoantigens for usein a vaccine or immunogenic composition. These problems may be addressedby:

-   -   identifying all, or nearly all, mutations in the neoplasia/tumor        at the DNA level using whole genome, whole exome (e.g., only        captured exons), or RNA sequencing of tumor versus matched        germline samples from each patient;    -   analyzing the identified mutations with one or more peptide-MHC        binding prediction algorithms to generate a plurality of        candidate neoantigen T cell epitopes that are expressed within        the neoplasia/tumor and may bind patient HLA alleles; and    -   synthesizing the plurality of candidate neoantigen peptides        selected from the sets of all neoORF peptides and predicted        binding peptides for use in a cancer vaccine or immunogenic        composition.

For example, translating sequencing information into a therapeuticvaccine may include:

(1) Prediction of Personal Mutated Peptides that can Bind to HLAMolecules of the Individual.

Efficiently choosing which particular mutations to utilize as immunogenrequires identification of the patient HLA type and the ability topredict which mutated peptides would efficiently bind to the patient'sHLA alleles. Recently, neural network based learning approaches withvalidated binding and non-binding peptides have advanced the accuracy ofprediction algorithms for the major HLA-A and -B alleles.

(2) Formulating the Drug as a Multi-Epitope Vaccine of Long Peptides.

Targeting as many mutated epitopes as practically possible takesadvantage of the enormous capacity of the immune system, prevents theopportunity for immunological escape by down-modulation of a particularimmune targeted gene product, and compensates for the known inaccuracyof epitope prediction approaches. Synthetic peptides provide aparticularly useful means to prepare multiple immunogens efficiently andto rapidly translate identification of mutant epitopes to an effectivevaccine. Peptides can be readily synthesized chemically and easilypurified utilizing reagents free of contaminating bacteria or animalsubstances. The small size allows a clear focus on the mutated region ofthe protein and also reduces irrelevant antigenic competition from othercomponents (unmutated protein or viral vector antigens).

(3) Combination with a Strong Vaccine Adjuvant.

Effective vaccines require a strong adjuvant to initiate an immuneresponse. As described below, poly-ICLC, an agonist of TLR3 and the RNAhelicase-domains of MDA5 and RIG3, has shown several desirableproperties for a vaccine adjuvant. These properties include theinduction of local and systemic activation of immune cells in vivo,production of stimulatory chemokines and cytokines, and stimulation ofantigen-presentation by DCs. Furthermore, poly-ICLC can induce durableCD4+ and CD8+ responses in humans. Importantly, striking similarities inthe upregulation of transcriptional and signal transduction pathwayswere seen in subjects vaccinated with poly-ICLC and in volunteers whohad received the highly effective, replication-competent yellow fevervaccine. Furthermore, >90% of ovarian carcinoma patients immunized withpoly-ICLC in combination with a NY-ES0-1 peptide vaccine (in addition toMontanide) showed induction of CD4+ and CD8+ T cell, as well as antibodyresponses to the peptide in a recent phase 1 study. At the same time,polyICLC has been extensively tested in more than 25 clinical trials todate and exhibited a relatively benign toxicity profile. The advantagesof the invention are described further herein.

As described herein, there is a large body of evidence in both animalsand humans that mutated epitopes are effective in inducing an immuneresponse and that cases of spontaneous tumor regression or long termsurvival correlate with CD8+ T-cell responses to mutated epitopes(Buckwalter and Srivastava P K. “It is the antigen(s), stupid” and otherlessons from over a decade of vaccitherapy of human cancer. Seminars inimmunology 20:296-300 (2008); Karanikas et al, High frequency ofcytolytic T lymphocytes directed against a tumor-specific mutatedantigen detectable with HLA tetramers in the blood of a lung carcinomapatient with long survival. Cancer Res. 61:3718-3724 (2001); Lennerz etal, The response of autologous T cells to a human melanoma is dominatedby mutated neoantigens. Proc Natl Acad Sci USA. 102:16013 (2005)) andthat “immunoediting” can be tracked to alterations in expression ofdominant mutated antigens in mice and man (Matsushita et al, Cancerexome analysis reveals a T-cell-dependent mechanism of cancerimmunoediting Nature 482:400 (2012); DuPage et al, Expression oftumor-specific antigens underlies cancer immunoediting Nature 482:405(2012); and Sampson et al, Immunologic escape after prolongedprogression-free survival with epidermal growth factor receptor variantIII peptide vaccination in patients with newly diagnosed glioblastoma JClin Oncol. 28:4722-4729 (2010)). In one embodiment, the mutatedepitopes of a cancer patient are determined.

In one embodiment mutated epitopes are determined by sequencing thegenome and/or exome of tumor tissue and healthy tissue from a cancerpatient using next generation sequencing technologies. In anotherembodiment genes that are selected based on their frequency of mutationand ability to act as a neoantigen are sequenced using next generationsequencing technology. Next-generation sequencing applies to genomesequencing, genome resequencing, transcriptome profiling (RNA-Seq),DNA-protein interactions (ChIP-sequencing), and epigenomecharacterization (de Magalhes J P, Finch C E, Janssens G (2010).“Next-generation sequencing in aging research: emerging applications,problems, pitfalls and possible solutions”. Ageing Research Reviews 9(3): 315-323; Hall N (May 2007). “Advanced sequencing technologies andtheir wider impact in microbiology”. J. Exp. Biol. 209 (Pt 9):1518-1525; Church G M (January 2006). “Genomes for all”. Sci. Am. 294(1): 46-54; ten Bosch J R, Grody W W (2008). “Keeping Up with the NextGeneration”. The Journal of Molecular Diagnostics 10 (6): 484-492;Tucker T, Marra M, Friedman J M (2009). “Massively Parallel Sequencing:The Next Big Thing in Genetic Medicine”. The American Journal of HumanGenetics 85 (2): 142-154). Next-generation sequencing can now rapidlyreveal the presence of discrete mutations such as coding mutations inindividual tumors, most commonly single amino acid changes (e.g.,missense mutations) and less frequently novel stretches of amino acidsgenerated by frame-shift insertions/deletions/gene fusions, read-throughmutations in stop codons, and translation of improperly spliced introns(e.g., neoORFs). NeoORFs are particularly valuable as immunogens becausethe entirety of their sequence is completely novel to the immune systemand so are analogous to a viral or bacterial foreign antigen. Thus,neoORFs: (1) are highly specific to the tumor (i.e. there is noexpression in any normal cells); and (2) can bypass central tolerance,thereby increasing the precursor frequency of neoantigen-specific CTLs.For example, the power of utilizing analogous foreign sequences in atherapeutic anti-cancer vaccine or immunogenic composition was recentlydemonstrated with peptides derived from human papilloma virus (HPV).˜50% of the 19 patients with pre-neoplastic, viral-induced disease whoreceived 3-4 vaccinations of a mix of HPV peptides derived from theviral oncogenes E6 and E7 maintained a complete response for ≥24 months(Kenter et a, Vaccination against HPV-16 Oncoproteins for VulvarIntraepithelial Neoplasia NEJM 361:1838 (2009)).

Sequencing technology has revealed that each tumor contains multiple,patient-specific mutations that alter the protein coding content of agene. Such mutations create altered proteins, ranging from single aminoacid changes (caused by missense mutations) to addition of long regionsof novel amino acid sequence due to frame shifts, read-through oftermination codons or translation of intron regions (novel open readingframe mutations; neoORFs). These mutated proteins are valuable targetsfor the host's immune response to the tumor as, unlike native proteins,they are not subject to the immune-dampening effects of self-tolerance.Therefore, mutated proteins are more likely to be immunogenic and arealso more specific for the tumor cells compared to normal cells of thepatient.

An alternative method for identifying tumor specific neoantigens isdirect protein sequencing. Protein sequencing of enzymatic digests usingmultidimensional MS techniques (MSn) including tandem mass spectrometry(MS/MS)) can also be used to identify neoantigens of the invention. Suchproteomic approaches permit rapid, highly automated analysis (see, e.g.,K. Gevaert and J. Vandekerckhove, Electrophoresis 21:1145-1154 (2000)).It is further contemplated within the scope of the invention thathigh-throughput methods for de novo sequencing of unknown proteins maybe used to analyze the proteome of a patient's tumor to identifyexpressed neoantigens. For example, meta shotgun protein sequencing maybe used to identify expressed neoantigens (see e.g., Guthals et al.(2012) Shotgun Protein Sequencing with Meta-contig Assembly, Molecularand Cellular Proteomics 11(10): 1084-96).

Tumor specific neoantigens may also be identified using MHC multimers toidentify neoantigen-specific T-cell responses. For example,high-throughput analysis of neoantigen-specific T-cell responses inpatient samples may be performed using MHC tetramer-based screeningtechniques (see e.g., Hombrink et al. (2011) High-ThroughputIdentification of Potential Minor Histocompatibility Antigens by MHCTetramer-Based Screening: Feasibility and Limitations 6(8): 1-11; Hadrupet al. (2009) Parallel detection of antigen-specific T-cell responses bymultidimensional encoding of MHC multimers, Nature Methods, 6(7):520-26;van Rooij et al. (2013) Tumor exome analysis reveals neoantigen-specificT-cell reactivity in an Ipilimumab-responsive melanoma, Journal ofClinical Oncology, 31:1-4; and Heemskerk et al. (2013) The cancerantigenome, EMBO Journal, 32(2):194-203). Such tetramer-based screeningtechniques may be used for the initial identification of tumor specificneoantigens, or alternatively as a secondary screening protocol toassess what neoantigens a patient may have already been exposed to,thereby facilitating the selection of candidate neoantigens for theinvention.

In one embodiment the sequencing data derived from determining thepresence of mutations in a cancer patient is analysed to predictpersonal mutated peptides that can bind to HLA molecules of theindividual. In one embodiment the data is analysed using a computer. Inanother embodiment the sequence data is analysed for the presence ofneoantigens. In one embodiment neoantigens are determined by theiraffinity to MHC molecules. Efficiently choosing which particularmutations to utilize as immunogen requires identification of the patientHLA type and the ability to predict which mutated peptides wouldefficiently bind to the patient's HLA alleles. Recently, neural networkbased learning approaches with validated binding and non-bindingpeptides have advanced the accuracy of prediction algorithms for themajor HLA-A and -B alleles. Utilizing the recently improved algorithmsfor predicting which missense mutations create strong binding peptidesto the patient's cognate MHC molecules, a set of peptides representativeof optimal mutated epitopes (both neoORF and missense) for each patientmay be identified and prioritized (Zhang et al, Machine learningcompetition in immunology—Prediction of HLA class I binding peptides JImmunol Methods 374:1 (2011); Lundegaard et al Prediction of epitopesusing neural network based methods J Immunol Methods 374:26 (2011)).

Targeting as many mutated epitopes as practically possible takesadvantage of the enormous capacity of the immune system, prevents theopportunity for immunological escape by down-modulation of a particularimmune targeted gene product, and compensates for the known inaccuracyof epitope prediction approaches. Synthetic peptides provide aparticularly useful means to prepare multiple immunogens efficiently andto rapidly translate identification of mutant epitopes to an effectivevaccine or immunogenic composition. Peptides can be readily synthesizedchemically and easily purified utilizing reagents free of contaminatingbacteria or animal substances. The small size allows a clear focus onthe mutated region of the protein and also reduces irrelevant antigeniccompetition from other components (unmutated protein or viral vectorantigens).

In one embodiment the drug formulation is a multi-epitope vaccine orimmunogenic composition of long peptides. Such “long” peptides undergoefficient internalization, processing and cross-presentation inprofessional antigen-presenting cells such as dendritic cells, and havebeen shown to induce CTLs in humans (Melief and van der Burg,Immunotherapy of established (pre) malignant disease by synthetic longpeptide vaccines Nature Rev Cancer 8:351 (2008)). In one embodiment atleast 1 peptide is prepared for immunization. In a preferred embodiment20 or more peptides are prepared for immunization. In one embodiment theneoantigenic peptide ranges from about 5 to about 50 amino acids inlength. In another embodiment peptides from about 15 to about 35 aminoacids in length is synthesized. In preferred embodiment the neoantigenicpeptide ranges from about 20 to about 35 amino acids in length.

Production of Tumor Specific Neoantigens

The present invention is based, at least in part, on the ability topresent the immune system of the patient with a pool of tumor specificneoantigens. One of skill in the art from this disclosure and theknowledge in the art will appreciate that there are a variety of ways inwhich to produce such tumor specific neoantigens. In general, such tumorspecific neoantigens may be produced either in vitro or in vivo. Tumorspecific neoantigens may be produced in vitro as peptides orpolypeptides, which may then be formulated into a personalized neoplasiavaccine or immunogenic composition and administered to a subject. Asdescribed in further detail herein, such in vitro production may occurby a variety of methods known to one of skill in the art such as, forexample, peptide synthesis or expression of a peptide/polypeptide from aDNA or RNA molecule in any of a variety of bacterial, eukaryotic, orviral recombinant expression systems, followed by purification of theexpressed peptide/polypeptide. Alternatively, tumor specific neoantigensmay be produced in vivo by introducing molecules (e.g., DNA, RNA, viralexpression systems, and the like) that encode tumor specific neoantigensinto a subject, whereupon the encoded tumor specific neoantigens areexpressed. The methods of in vitro and in vivo production of neoantigensis also further described herein as it relates to pharmaceuticalcompositions and methods of delivery.

Selection of Peptides Soluble in an Aqueous Solution

The methods disclosed herein are based, at least in part, on the abilityto select peptides that are soluble in an aqueous solution. Solubilityof peptides may be determined experimentally. The solubility of peptidesin an aqueous solution can also be determined based on the amino acidsequence of each peptide. In one embodiment, the solubility of a peptideis determined using two calculable parameters that relate tohydrophobicity and the isoelectric point (Pi) of the peptide.Isoelectric point and hydrophobicity can be estimated using any of themethods known to one of skill, for example, the methods described inExample 14. In one embodiment, hydrophobicity of a peptide is estimatedby identifying regions within the peptide that consists of consecutivehydrophobic amino acids, calculating an index for the degree ofhydrophobicity of each region of consecutive hydrophobic amino acids,and identifying the region with the highest degree of hydrophobicity.This parameter can be designated HYDRO. This calculation can be readilyaccomplished by using published values of hydrophobicity (orhydrophilicity) for each amino acid side chain, identifyinguninterrupted stretches of hydrophobic amino acids in the peptide andsumming the hydrophobicity of each amino acid in each region. An examplefor estimating the hydrophobicity of a peptide is described in Example14.

In one embodiment, a method of selecting a soluble peptide describedherein comprises determining the Pi and HYDRO value of a peptide andselecting the peptide when its Pi and HYDRO is bounded by Pi ≥5 andHYDRO 2-6.0, Pi ≥8 and HYDRO ≥−8.0, Pi ≤5 and HYDRO ≥−5, and Pi ≥9 andHYDRO ≤−8.0.

In one embodiment, a method of assessing the solubility of a peptide inan aqueous solution described herein comprises determining theisoelectric point (Pi) and hydrophobicity (HYDRO) of the peptide,wherein the peptide is soluble in the aqueous solution when its Pi andHYDRO is bounded by Pi ≥5 and HYDRO 2-6.0, Pi ≥8 and HYDRO ≥−8.0, Pi ≤5and HYDRO ≥−5, and Pi ≥9 and HYDRO ≤−8.0.

In one embodiment, a method of preparing an aqueous peptide solutiondescribed herein comprises determining the isoelectric point (Pi) andhydrophobicity (HYDRO) of at least one peptide, selecting the peptidewhen its Pi and HYDRO is bounded by Pi ≥5 and HYDRO ≥−6.0, Pi ≥8 andHYDRO ≥−8.0, Pi ≤5 and HYDRO ≥−5, and Pi ≥9 and HYDRO ≤−8.0, andpreparing an aqueous solution comprising the peptide.

In one embodiment, a method of preparing an aqueous neo-antigenicpeptide solution described herein comprises determining the isoelectricpoint (Pi) and hydrophobicity (HYDRO) of at least one neo-antigenicpeptide, selecting the at least one neo-antigenic peptide if its Pi andHYDRO is bounded by Pi ≥5 and HYDRO 2-6.0, Pi ≥8 and HYDRO ≥−8.0, Pi ≤5and HYDRO ≥−5, and Pi ≥9 and HYDRO ≤−8.0, preparing a solutioncomprising the at least one neo-antigenic peptide or a pharmaceuticallyacceptable salt thereof, and combining the solution comprising the atleast one neo-antigenic peptide or a pharmaceutically acceptable saltthereof with a solution comprising succinic acid or a pharmaceuticallyacceptable salt thereof, thereby preparing a peptide solution for aneoplasia vaccine.

In Vitro Peptide/Polypeptide Synthesis

Proteins or peptides may be made by any technique known to those ofskill in the art, including the expression of proteins, polypeptides orpeptides through standard molecular biological techniques, the isolationof proteins or peptides from natural sources, in vitro translation, orthe chemical synthesis of proteins or peptides. The nucleotide andprotein, polypeptide and peptide sequences corresponding to variousgenes have been previously disclosed, and may be found at computerizeddatabases known to those of ordinary skill in the art. One such databaseis the National Center for Biotechnology Information's Genbank andGenPept databases located at the National Institutes of Health website.The coding regions for known genes may be amplified and/or expressedusing the techniques disclosed herein or as would be known to those ofordinary skill in the art. Alternatively, various commercialpreparations of proteins, polypeptides and peptides are known to thoseof skill in the art.

Peptides can be readily synthesized chemically utilizing reagents thatare free of contaminating bacterial or animal substances (Merrifield RB: Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J.Am. Chem. Soc. 85:2149-54, 1963). In certain embodiments, neoantigenicpeptides are prepared by (1) parallel solid-phase synthesis onmulti-channel instruments using uniform synthesis and cleavageconditions; (2) purification over a RP-HPLC column with columnstripping; and re-washing, but not replacement, between peptides;followed by (3) analysis with a limited set of the most informativeassays. The Good Manufacturing Practices (GMP) footprint can be definedaround the set of peptides for an individual patient, thus requiringsuite changeover procedures only between syntheses of peptides fordifferent patients.

Alternatively, a nucleic acid (e.g., a polynucleotide) encoding aneoantigenic peptide of the invention may be used to produce theneoantigenic peptide in vitro. The polynucleotide may be, e.g., DNA,cDNA, PNA, CNA, RNA, either single- and/or double-stranded, or native orstabilized forms of polynucleotides, such as e.g. polynucleotides with aphosphorothiate backbone, or combinations thereof and it may or may notcontain introns so long as it codes for the peptide. In one embodimentin vitro translation is used to produce the peptide. Many exemplarysystems exist that one skilled in the art could utilize (e.g., ReticLysate IVT Kit, Life Technologies, Waltham, Mass.).

An expression vector capable of expressing a polypeptide can also beprepared. Expression vectors for different cell types are well known inthe art and can be selected without undue experimentation. Generally,the DNA is inserted into an expression vector, such as a plasmid, inproper orientation and correct reading frame for expression. Ifnecessary, the DNA may be linked to the appropriate transcriptional andtranslational regulatory control nucleotide sequences recognized by thedesired host (e.g., bacteria), although such controls are generallyavailable in the expression vector. The vector is then introduced intothe host bacteria for cloning using standard techniques (see, e.g.,Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Expression vectors comprising the isolated polynucleotides, as well ashost cells containing the expression vectors, are also contemplated. Theneoantigenic peptides may be provided in the form of RNA or cDNAmolecules encoding the desired neoantigenic peptides. One or moreneoantigenic peptides of the invention may be encoded by a singleexpression vector.

The term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequences for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequences. Polynucleotides can be in the form of RNA or inthe form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; andcan be double-stranded or single-stranded, and if single stranded can bethe coding strand or non-coding (anti-sense) strand.

In embodiments, the polynucleotides may comprise the coding sequence forthe tumor specific neoantigenic peptide fused in the same reading frameto a polynucleotide which aids, for example, in expression and/orsecretion of a polypeptide from a host cell (e.g., a leader sequencewhich functions as a secretory sequence for controlling transport of apolypeptide from the cell). The polypeptide having a leader sequence isa preprotein and can have the leader sequence cleaved by the host cellto form the mature form of the polypeptide.

In embodiments, the polynucleotides can comprise the coding sequence forthe tumor specific neoantigenic peptide fused in the same reading frameto a marker sequence that allows, for example, for purification of theencoded polypeptide, which may then be incorporated into thepersonalized neoplasia vaccine or immunogenic composition. For example,the marker sequence can be a hexa-histidine tag supplied by a pQE-9vector to provide for purification of the mature polypeptide fused tothe marker in the case of a bacterial host, or the marker sequence canbe a hemagglutinin (HA) tag derived from the influenza hemagglutininprotein when a mammalian host (e.g., COS-7 cells) is used. Additionaltags include, but are not limited to, Calmodulin tags, FLAG tags, Myctags, S tags, SBP tags, Softag 1, Softag 3, V5 tag, Xpress tag,Isopeptag, SpyTag, Biotin Carboxyl Carrier Protein (BCCP) tags, GSTtags, fluorescent protein tags (e.g., green fluorescent protein tags),maltose binding protein tags, Nus tags, Strep-tag, thioredoxin tag, TCtag, Ty tag, and the like.

In embodiments, the polynucleotides may comprise the coding sequence forone or more of the tumor specific neoantigenic peptides fused in thesame reading frame to create a single concatamerized neoantigenicpeptide construct capable of producing multiple neoantigenic peptides.

In certain embodiments, isolated nucleic acid molecules having anucleotide sequence at least 60% identical, at least 65% identical, atleast 70% identical, at least 75% identical, at least 80% identical, atleast 85% identical, at least 90% identical, at least 95% identical, orat least 96%, 97%, 98% or 99% identical to a polynucleotide encoding atumor specific neoantigenic peptide of the present invention, can beprovided.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence can include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence. In other words, to obtain a polynucleotide having a nucleotidesequence at least 95% identical to a reference nucleotide sequence, upto 5% of the nucleotides in the reference sequence can be deleted orsubstituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence can be inserted intothe reference sequence. These mutations of the reference sequence canoccur at the amino- or carboxy-terminal positions of the referencenucleotide sequence or anywhere between those terminal positions,interspersed either individually among nucleotides in the referencesequence or in one or more contiguous groups within the referencesequence.

As a practical matter, whether any particular nucleic acid molecule isat least 80% identical, at least 85% identical, at least 90% identical,and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% identicalto a reference sequence can be determined conventionally using knowncomputer programs such as the Bestfit program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).Bestfit uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2:482-489 (1981), to find the bestsegment of homology between two sequences. When using Bestfit or anyother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set such that thepercentage of identity is calculated over the full length of thereference nucleotide sequence and that gaps in homology of up to 5% ofthe total number of nucleotides in the reference sequence are allowed.

The isolated tumor specific neoantigenic peptides described herein canbe produced in vitro (e.g., in the laboratory) by any suitable methodknown in the art. Such methods range from direct protein syntheticmethods to constructing a DNA sequence encoding isolated polypeptidesequences and expressing those sequences in a suitable transformed host.In some embodiments, a DNA sequence is constructed using recombinanttechnology by isolating or synthesizing a DNA sequence encoding awild-type protein of interest. Optionally, the sequence can bemutagenized by site-specific mutagenesis to provide functional analogsthereof. See, e.g. Zoeller et al., Proc. Nat'l. Acad. Sci. USA81:5662-5066 (1984) and U.S. Pat. No. 4,588,585.

In embodiments, a DNA sequence encoding a polypeptide of interest wouldbe constructed by chemical synthesis using an oligonucleotidesynthesizer. Such oligonucleotides can be designed based on the aminoacid sequence of the desired polypeptide and selecting those codons thatare favored in the host cell in which the recombinant polypeptide ofinterest is produced. Standard methods can be applied to synthesize anisolated polynucleotide sequence encoding an isolated polypeptide ofinterest. For example, a complete amino acid sequence can be used toconstruct a back-translated gene. Further, a DNA oligomer containing anucleotide sequence coding for the particular isolated polypeptide canbe synthesized. For example, several small oligonucleotides coding forportions of the desired polypeptide can be synthesized and then ligated.The individual oligonucleotides typically contain 5′ or 3′ overhangs forcomplementary assembly.

Once assembled (e.g., by synthesis, site-directed mutagenesis, oranother method), the polynucleotide sequences encoding a particularisolated polypeptide of interest is inserted into an expression vectorand optionally operatively linked to an expression control sequenceappropriate for expression of the protein in a desired host. Properassembly can be confirmed by nucleotide sequencing, restriction mapping,and expression of a biologically active polypeptide in a suitable host.As well known in the art, in order to obtain high expression levels of atransfected gene in a host, the gene can be operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host.

Recombinant expression vectors may be used to amplify and express DNAencoding the tumor specific neoantigenic peptides. Recombinantexpression vectors are replicable DNA constructs which have synthetic orcDNA-derived DNA fragments encoding a tumor specific neoantigenicpeptide or a bioequivalent analog operatively linked to suitabletranscriptional or translational regulatory elements derived frommammalian, microbial, viral or insect genes. A transcriptional unitgenerally comprises an assembly of (1) a genetic element or elementshaving a regulatory role in gene expression, for example,transcriptional promoters or enhancers, (2) a structural or codingsequence which is transcribed into mRNA and translated into protein, and(3) appropriate transcription and translation initiation and terminationsequences, as described in detail herein. Such regulatory elements caninclude an operator sequence to control transcription. The ability toreplicate in a host, usually conferred by an origin of replication, anda selection gene to facilitate recognition of transformants canadditionally be incorporated. DNA regions are operatively linked whenthey are functionally related to each other. For example, DNA for asignal peptide (secretory leader) is operatively linked to DNA for apolypeptide if it is expressed as a precursor which participates in thesecretion of the polypeptide; a promoter is operatively linked to acoding sequence if it controls the transcription of the sequence; or aribosome binding site is operatively linked to a coding sequence if itis positioned so as to permit translation. Generally, operatively linkedmeans contiguous, and in the case of secretory leaders, means contiguousand in reading frame. Structural elements intended for use in yeastexpression systems include a leader sequence enabling extracellularsecretion of translated protein by a host cell. Alternatively, whererecombinant protein is expressed without a leader or transport sequence,it can include an N-terminal methionine residue. This residue canoptionally be subsequently cleaved from the expressed recombinantprotein to provide a final product.

Useful expression vectors for eukaryotic hosts, especially mammals orhumans include, for example, vectors comprising expression controlsequences from SV40, bovine papilloma virus, adenovirus andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from Escherichia coli,including pCR 1, pBR322, pMB9 and their derivatives, wider host rangeplasmids, such as M13 and filamentous single-stranded DNA phages.

Suitable host cells for expression of a polypeptide include prokaryotes,yeast, insect or higher eukaryotic cells under the control ofappropriate promoters. Prokaryotes include gram negative or grampositive organisms, for example E. coli or bacilli. Higher eukaryoticcells include established cell lines of mammalian origin. Cell-freetranslation systems could also be employed. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are well known in the art (see Pouwels et al., CloningVectors: A Laboratory Manual, Elsevier, N.Y., 1985).

Various mammalian or insect cell culture systems are also advantageouslyemployed to express recombinant protein. Expression of recombinantproteins in mammalian cells can be performed because such proteins aregenerally correctly folded, appropriately modified and completelyfunctional. Examples of suitable mammalian host cell lines include theCOS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175,1981), and other cell lines capable of expressing an appropriate vectorincluding, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO),293, HeLa and BHK cell lines. Mammalian expression vectors can comprisenontranscribed elements such as an origin of replication, a suitablepromoter and enhancer linked to the gene to be expressed, and other 5′or 3′ flanking nontranscribed sequences, and 5′ or 3′ nontranslatedsequences, such as necessary ribosome binding sites, a polyadenylationsite, splice donor and acceptor sites, and transcriptional terminationsequences. Baculovirus systems for production of heterologous proteinsin insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47(1988).

The proteins produced by a transformed host can be purified according toany suitable method. Such standard methods include chromatography (e.g.,ion exchange, affinity and sizing column chromatography, and the like),centrifugation, differential solubility, or by any other standardtechnique for protein purification. Affinity tags such as hexahistidine,maltose binding domain, influenza coat sequence,glutathione-S-transferase, and the like can be attached to the proteinto allow easy purification by passage over an appropriate affinitycolumn. Isolated proteins can also be physically characterized usingsuch techniques as proteolysis, nuclear magnetic resonance and x-raycrystallography.

For example, supernatants from systems which secrete recombinant proteininto culture media can be first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. Following the concentrationstep, the concentrate can be applied to a suitable purification matrix.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a cancer stem cell protein-Fc composition.Some or all of the foregoing purification steps, in variouscombinations, can also be employed to provide a homogeneous recombinantprotein.

Recombinant protein produced in bacterial culture can be isolated, forexample, by initial extraction from cell pellets, followed by one ormore concentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. High performance liquid chromatography (HPLC) canbe employed for final purification steps. Microbial cells employed inexpression of a recombinant protein can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

In Vivo Peptide/Polypeptide Synthesis

The present invention also contemplates the use of nucleic acidmolecules as vehicles for delivering neoantigenic peptides/polypeptidesto the subject in need thereof, in vivo, in the form of, e.g., DNA/RNAvaccines (see, e.g., WO2012/159643, and WO2012/159754, herebyincorporated by reference in their entirety).

In one embodiment neoantigens may be administered to a patient in needthereof by use of a plasmid. These are plasmids which usually consist ofa strong viral promoter to drive the in vivo transcription andtranslation of the gene (or complementary DNA) of interest (Mor, et al.,(1995). The Journal of Immunology 155 (4): 2039-2046). Intron A maysometimes be included to improve mRNA stability and hence increaseprotein expression (Leitner et al. (1997). The Journal of Immunology 159(12): 6112-6119). Plasmids also include a strongpolyadenylation/transcriptional termination signal, such as bovinegrowth hormone or rabbit beta-globulin polyadenylation sequences(Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42:343-410; Robinson et al., (2000). Adv. Virus Res. Advances in VirusResearch 55: 1-74; Böhm et al., (1996). Journal of Immunological Methods193 (1): 29-40.). Multicistronic vectors are sometimes constructed toexpress more than one immunogen, or to express an immunogen and animmunostimulatory protein (Lewis et al., (1999). Advances in VirusResearch (Academic Press) 54: 129-88).

Because the plasmid is the “vehicle” from which the immunogen isexpressed, optimising vector design for maximal protein expression isessential (Lewis et al., (1999). Advances in Virus Research (AcademicPress) 54: 129-88). One way of enhancing protein expression is byoptimising the codon usage of pathogenic mRNAs for eukaryotic cells.Another consideration is the choice of promoter. Such promoters may bethe SV40 promoter or Rous Sarcoma Virus (RSV).

Plasmids may be introduced into animal tissues by a number of differentmethods. The two most popular approaches are injection of DNA in saline,using a standard hypodermic needle, and gene gun delivery. A schematicoutline of the construction of a DNA vaccine plasmid and its subsequentdelivery by these two methods into a host is illustrated at ScientificAmerican (Weiner et al., (1999) Scientific American 281 (1): 34-41).Injection in saline is normally conducted intramuscularly (IM) inskeletal muscle, or intradermally (ID), with DNA being delivered to theextracellular spaces. This can be assisted by electroporation bytemporarily damaging muscle fibres with myotoxins such as bupivacaine;or by using hypertonic solutions of saline or sucrose (Alarcon et al.,(1999). Adv. Parasitol. Advances in Parasitology 42: 343-410). Immuneresponses to this method of delivery can be affected by many factors,including needle type, needle alignment, speed of injection, volume ofinjection, muscle type, and age, sex and physiological condition of theanimal being injected (Alarcon et al., (1999). Adv. Parasitol. Advancesin Parasitology 42: 343-410).

Gene gun delivery, the other commonly used method of delivery,ballistically accelerates plasmid DNA (pDNA) that has been adsorbed ontogold or tungsten microparticles into the target cells, using compressedhelium as an accelerant (Alarcon et al., (1999). Adv. Parasitol.Advances in Parasitology 42: 343-410; Lewis et al., (1999). Advances inVirus Research (Academic Press) 54: 129-88).

Alternative delivery methods may include aerosol instillation of nakedDNA on mucosal surfaces, such as the nasal and lung mucosa, (Lewis etal., (1999). Advances in Virus Research (Academic Press) 54: 129-88) andtopical administration of pDNA to the eye and vaginal mucosa (Lewis etal., (1999) Advances in Virus Research (Academic Press) 54: 129-88).Mucosal surface delivery has also been achieved using cationicliposome-DNA preparations, biodegradable microspheres, attenuatedShigella or Listeria vectors for oral administration to the intestinalmucosa, and recombinant adenovirus vectors.

The method of delivery determines the dose of DNA required to raise aneffective immune response. Saline injections require variable amounts ofDNA, from 10 μg-1 mg, whereas gene gun deliveries require 100 to 1000times less DNA than intramuscular saline injection to raise an effectiveimmune response. Generally, 0.2 μg-20 μg are required, althoughquantities as low as 16 ng have been reported. These quantities varyfrom species to species, with mice, for example, requiring approximately10 times less DNA than primates. Saline injections require more DNAbecause the DNA is delivered to the extracellular spaces of the targettissue (normally muscle), where it has to overcome physical barriers(such as the basal lamina and large amounts of connective tissue, tomention a few) before it is taken up by the cells, while gene gundeliveries bombard DNA directly into the cells, resulting in less“wastage” (See e.g., Sedegah et al., (1994). Proceedings of the NationalAcademy of Sciences of the United States of America 91 (21): 9866-9870;Daheshia et al., (1997). The Journal of Immunology 159 (4): 1945-1952;Chen et al., (1998). The Journal of Immunology 160 (5): 2425-2432;Sizemore (1995) Science 270 (5234): 299-302; Fynan et al., (1993) Proc.Natl. Acad. Sci. U.S.A. 90 (24): 11478-82).

In one embodiment, a neoplasia vaccine or immunogenic composition mayinclude separate DNA plasmids encoding, for example, one or moreneoantigenic peptides/polypeptides as identified in according to theinvention. As discussed herein, the exact choice of expression vectorscan depend upon the peptide/polypeptides to be expressed, and is wellwithin the skill of the ordinary artisan. The expected persistence ofthe DNA constructs (e.g., in an episomal, non-replicating,non-integrated form in the muscle cells) is expected to provide anincreased duration of protection.

One or more neoantigenic peptides of the invention may be encoded andexpressed in vivo using a viral based system (e.g., an adenovirussystem, an adeno associated virus (AAV) vector, a poxvirus, or alentivirus). In one embodiment, the neoplasia vaccine or immunogeniccomposition may include a viral based vector for use in a human patientin need thereof, such as, for example, an adenovirus (see, e.g., Badenet al. First-in-human evaluation of the safety and immunogenicity of arecombinant adenovirus serotype 26 HIV-1 Env vaccine (IPCAVD 001). JInfect Dis. 2013 Jan. 15; 207(2):240-7, hereby incorporated by referencein its entirety). Plasmids that can be used for adeno associated virus,adenovirus, and lentivirus delivery have been described previously (seee.g., U.S. Pat. Nos. 6,955,808 and 6,943,019, and U.S. Patentapplication No. 20080254008, hereby incorporated by reference).

Among vectors that may be used in the practice of the invention,integration in the host genome of a cell is possible with retrovirusgene transfer methods, often resulting in long term expression of theinserted transgene. In a preferred embodiment the retrovirus is alentivirus. Additionally, high transduction efficiencies have beenobserved in many different cell types and target tissues. The tropism ofa retrovirus can be altered by incorporating foreign envelope proteins,expanding the potential target population of target cells. A retroviruscan also be engineered to allow for conditional expression of theinserted transgene, such that only certain cell types are infected bythe lentivirus. Cell type specific promoters can be used to targetexpression in specific cell types. Lentiviral vectors are retroviralvectors (and hence both lentiviral and retroviral vectors may be used inthe practice of the invention). Moreover, lentiviral vectors arepreferred as they are able to transduce or infect non-dividing cells andtypically produce high viral titers. Selection of a retroviral genetransfer system may therefore depend on the target tissue. Retroviralvectors are comprised of cis-acting long terminal repeats with packagingcapacity for up to 6-10 kb of foreign sequence. The minimum cis-actingLTRs are sufficient for replication and packaging of the vectors, whichare then used to integrate the desired nucleic acid into the target cellto provide permanent expression. Widely used retroviral vectors that maybe used in the practice of the invention include those based upon murineleukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immunodeficiency virus (SIV), human immuno deficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., (1992) J. Virol.66:2731-2739; Johann et al., (1992) J. Virol. 66:1635-1640; Sommnerfeltet al., (1990) Virol. 176:58-59; Wilson et al., (1998) J. Virol.63:2374-2378; Miller et al., (1991) J. Virol. 65:2220-2224;PCT/US94/05700). Zou et al. administered about 10 μl of a recombinantlentivirus having a titer of 1×10⁹ transducing units (TU)/ml by anintrathecal catheter. These sort of dosages can be adapted orextrapolated to use of a retroviral or lentiviral vector in the presentinvention.

Also useful in the practice of the invention is a minimal non-primatelentiviral vector, such as a lentiviral vector based on the equineinfectious anemia virus (EIAV) (see, e.g., Balagaan, (2006) J Gene Med;8: 275-285, Published online 21 Nov. 2005 in Wiley InterScience(www.interscience.wiley.com). DOI: 10.1002/jgm.845). The vectors mayhave cytomegalovirus (CMV) promoter driving expression of the targetgene. Accordingly, the invention contemplates amongst vector(s) usefulin the practice of the invention: viral vectors, including retroviralvectors and lentiviral vectors.

Also useful in the practice of the invention is an adenovirus vector.One advantage is the ability of recombinant adenoviruses to efficientlytransfer and express recombinant genes in a variety of mammalian cellsand tissues in vitro and in vivo, resulting in the high expression ofthe transferred nucleic acids. Further, the ability to productivelyinfect quiescent cells, expands the utility of recombinant adenoviralvectors. In addition, high expression levels ensure that the products ofthe nucleic acids will be expressed to sufficient levels to generate animmune response (see e.g., U.S. Pat. No. 7,029,848, hereby incorporatedby reference).

In an embodiment herein the delivery is via an adenovirus, which may beat a single booster dose containing at least 1×10⁵ particles (alsoreferred to as particle units, pu) of adenoviral vector. In anembodiment herein, the dose preferably is at least about 1×10⁶ particles(for example, about 1×10⁶-1×10¹² particles), more preferably at leastabout 1×10⁷ particles, more preferably at least about 1×10⁸ particles(e.g., about 1×10⁸-1×10¹¹ particles or about 1×10⁸-1×10¹² particles),and most preferably at least about 1×10⁹ particles (e.g., about1×10⁹-1×10¹⁰ particles or about 1×10⁹-1×10¹² particles), or even atleast about 1×10¹⁰ particles (e.g., about 1×10¹⁰-1×10¹² particles) ofthe adenoviral vector. Alternatively, the dose comprises no more thanabout 1×10¹⁴ particles, preferably no more than about 1×10¹³ particles,even more preferably no more than about 1×10¹² particles, even morepreferably no more than about 1×10¹¹ particles, and most preferably nomore than about 1×10¹⁰ particles (e.g., no more than about 1×10⁹articles). Thus, the dose may contain a single dose of adenoviral vectorwith, for example, about 1×10⁶ particle units (pu), about 2×10⁶ pu,about 4×10⁶ pu, about 1×10⁷ pu, about 2×10⁷ pu, about 4×10⁷ pu, about1×10⁸ pu, about 2×10⁸ pu, about 4×10⁸ pu, about 1×10⁹ pu, about 2×10⁹pu, about 4×10⁹ pu, about 1×10¹⁰ pu, about 2×10¹⁰ pu, about 4×10¹⁰ pu,about 1×10¹¹ pu, about 2×10¹¹ pu, about 4×10¹¹ pu, about 1×10¹² pu,about 2×10¹² pu, or about 4×10¹² pu of adenoviral vector. See, forexample, the adenoviral vectors in U.S. Pat. No. 8,454,972 B2 to Nabel,et. al., granted on Jun. 4, 2013; incorporated by reference herein, andthe dosages at col 29, lines 36-58 thereof. In an embodiment herein, theadenovirus is delivered via multiple doses.

In terms of in vivo delivery, AAV is advantageous over other viralvectors due to low toxicity and low probability of causing insertionalmutagenesis because it doesn't integrate into the host genome. AAV has apackaging limit of 4.5 or 4.75 Kb. Constructs larger than 4.5 or 4.75 Kbresult in significantly reduced virus production. There are manypromoters that can be used to drive nucleic acid molecule expression.AAV ITR can serve as a promoter and is advantageous for eliminating theneed for an additional promoter element. For ubiquitous expression, thefollowing promoters can be used: CMV, CAG, CBh, PGK, SV40, Ferritinheavy or light chains, etc. For brain expression, the followingpromoters can be used: SynapsinI for all neurons, CaMKIIalpha forexcitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc.Promoters used to drive RNA synthesis can include: Pol III promoterssuch as U6 or H1. The use of a Pol II promoter and intronic cassettescan be used to express guide RNA (gRNA).

As to AAV, the AAV can be AAV1, AAV2, AAV5 or any combination thereof.One can select the AAV with regard to the cells to be targeted; e.g.,one can select AAV serotypes 1, 2, 5 or a hybrid capsid AAV1, AAV2, AAV5or any combination thereof for targeting brain or neuronal cells; andone can select AAV4 for targeting cardiac tissue. AAV8 is useful fordelivery to the liver. The above promoters and vectors are preferredindividually.

In an embodiment herein, the delivery is via an AAV. A therapeuticallyeffective dosage for in vivo delivery of the AAV to a human is believedto be in the range of from about 20 to about 50 ml of saline solutioncontaining from about 1×10¹⁰ to about 1×10⁵⁰ functional AAV/ml solution.The dosage may be adjusted to balance the therapeutic benefit againstany side effects. In an embodiment herein, the AAV dose is generally inthe range of concentrations of from about 1×10⁵ to 1×10⁵⁰ genomes AAV,from about 1×10⁸ to 1×10²⁰ genomes AAV, from about 1×10¹⁰ to about1×10¹⁶ genomes, or about 1×10¹¹ to about 1×10¹⁶ genomes AAV. A humandosage may be about 1×10¹³ genomes AAV. Such concentrations may bedelivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50ml, or about 10 to about 25 ml of a carrier solution. In a preferredembodiment, AAV is used with a titer of about 2×10¹³ viralgenomes/milliliter, and each of the striatal hemispheres of a mousereceives one 500 nanoliter injection. Other effective dosages can bereadily established by one of ordinary skill in the art through routinetrials establishing dose response curves. See, for example, U.S. Pat.No. 8,404,658 B2 to Hajjar, et al., granted on Mar. 26, 2013, at col.27, lines 45-60.

In another embodiment effectively activating a cellular immune responsefor a neoplasia vaccine or immunogenic composition can be achieved byexpressing the relevant neoantigens in a vaccine or immunogeniccomposition in a non-pathogenic microorganism. Well-known examples ofsuch microorganisms are Mycobacterium bovis BCG, Salmonella andPseudomona (See, U.S. Pat. No. 6,991,797, hereby incorporated byreference in its entirety).

In another embodiment a Poxvirus is used in the neoplasia vaccine orimmunogenic composition. These include orthopoxvirus, avipox, vaccinia,MVA, NYVAC, canarypox, ALVAC, fowlpox, TROVAC, etc. (see e.g., Verardiet al., Hum Vaccin Immunother. 2012 July; 8(7):961-70; and Moss,Vaccine. 2013; 31(39): 4220-4222). Poxvirus expression vectors weredescribed in 1982 and quickly became widely used for vaccine developmentas well as research in numerous fields. Advantages of the vectorsinclude simple construction, ability to accommodate large amounts offoreign DNA and high expression levels.

In another embodiment the vaccinia virus is used in the neoplasiavaccine or immunogenic composition to express a neoantigen. (Rolph etal., Recombinant viruses as vaccines and immunological tools. Curr OpinImmunol 9:517-524, 1997). The recombinant vaccinia virus is able toreplicate within the cytoplasm of the infected host cell and thepolypeptide of interest can therefore induce an immune response.Moreover, Poxviruses have been widely used as vaccine or immunogeniccomposition vectors because of their ability to target encoded antigensfor processing by the major histocompatibility complex class I pathwayby directly infecting immune cells, in particular antigen-presentingcells, but also due to their ability to self-adjuvant.

In another embodiment ALVAC is used as a vector in a neoplasia vaccineor immunogenic composition. ALVAC is a canarypox virus that can bemodified to express foreign transgenes and has been used as a method forvaccination against both prokaryotic and eukaryotic antigens (Horig H,Lee D S, Conkright W, et al. Phase I clinical trial of a recombinantcanarypoxvirus (ALVAC) vaccine expressing human carcinoembryonic antigenand the B7.1 costimulatory molecule. Cancer Immunol Immunother 2000;49:504-14; von Mehren M, Arlen P, Tsang K Y, et al. Pilot study of adual gene recombinant avipox vaccine containing both carcinoembryonicantigen (CEA) and B7.1 transgenes in patients with recurrentCEA-expressing adenocarcinomas. Clin Cancer Res 2000; 6:2219-28; MuseyL, Ding Y, Elizaga M, et al. HIV-1 vaccination administeredintramuscularly can induce both systemic and mucosal T cell immunity inHIV-1-uninfected individuals. J Immunol 2003; 171:1094-101; Paoletti E.Applications of pox virus vectors to vaccination: an update. Proc NatlAcad Sci USA 1996; 93:11349-53; U.S. Pat. No. 7,255,862). In a phase Iclinical trial, an ALVAC virus expressing the tumor antigen CEA showedan excellent safety profile and resulted in increased CEA-specificT-cell responses in selected patients; objective clinical responses,however, were not observed (Marshall J L, Hawkins M J, Tsang K Y, et al.Phase I study in cancer patients of a replication-defective avipoxrecombinant vaccine that expresses human carcinoembryonic antigen. JClin Oncol 1999; 17:332-7).

In another embodiment a Modified Vaccinia Ankara (MVA) virus may be usedas a viral vector for a neoantigen vaccine or immunogenic composition.MVA is a member of the Orthopoxvirus family and has been generated byabout 570 serial passages on chicken embryo fibroblasts of the Ankarastrain of Vaccinia virus (CVA) (for review see Mayr, A., et al.,Infection 3, 6-14, 1975). As a consequence of these passages, theresulting MVA virus contains 31 kilobases less genomic informationcompared to CVA, and is highly host-cell restricted (Meyer, H. et al.,J. Gen. Virol. 72, 1031-1038, 1991). MVA is characterized by its extremeattenuation, namely, by a diminished virulence or infectious ability,but still holds an excellent immunogenicity. When tested in a variety ofanimal models, MVA was proven to be avirulent, even in immuno-suppressedindividuals. Moreover, MVA-BN®-HER2 is a candidate immunotherapydesigned for the treatment of HER-2-positive breast cancer and iscurrently in clinical trials. (Mandl et al., Cancer Immunol Immunother.January 2012; 61(1): 19-29). Methods to make and use recombinant MVA hasbeen described (e.g., see U.S. Pat. Nos. 8,309,098 and 5,185,146 herebyincorporated in its entirety).

In another embodiment the modified Copenhagen strain of vaccinia virus,NYVAC and NYVAC variations are used as a vector (see U.S. Pat. No.7,255,862; PCT WO 95/30018; U.S. Pat. Nos. 5,364,773 and 5,494,807,hereby incorporated by reference in its entirety).

In one embodiment recombinant viral particles of the vaccine orimmunogenic composition are administered to patients in need thereof.Dosages of expressed neoantigen can range from a few to a few hundredmicrograms, e.g., 5 to 500 .mu.g. The vaccine or immunogenic compositioncan be administered in any suitable amount to achieve expression atthese dosage levels. The viral particles can be administered to apatient in need thereof or transfected into cells in an amount of aboutat least 10^(3.5) pfu; thus, the viral particles are preferablyadministered to a patient in need thereof or infected or transfectedinto cells in at least about 10⁴ pfu to about 10⁶ pfu; however, apatient in need thereof can be administered at least about 10⁸ pfu suchthat a more preferred amount for administration can be at least about10⁷ pfu to about 10⁹ pfu. Doses as to NYVAC are applicable as to ALVAC,MVA, MVA-BN, and avipoxes, such as canarypox and fowlpox.

Vaccine or Immunogenic Composition Adjuvant

Effective vaccine or immunogenic compositions advantageously include astrong adjuvant to initiate an immune response. As described herein,poly-ICLC, an agonist of TLR3 and the RNA helicase-domains of MDA5 andRIG3, has shown several desirable properties for a vaccine orimmunogenic composition adjuvant. These properties include the inductionof local and systemic activation of immune cells in vivo, production ofstimulatory chemokines and cytokines, and stimulation ofantigen-presentation by DCs. Furthermore, poly-ICLC can induce durableCD4+ and CD8+ responses in humans. Importantly, striking similarities inthe upregulation of transcriptional and signal transduction pathwayswere seen in subjects vaccinated with poly-ICLC and in volunteers whohad received the highly effective, replication-competent yellow fevervaccine. Furthermore, >90% of ovarian carcinoma patients immunized withpoly-ICLC in combination with a NY-ESO-1 peptide vaccine (in addition toMontanide) showed induction of CD4+ and CD8+ T cell, as well as antibodyresponses to the peptide in a recent phase 1 study. At the same time,poly-ICLC has been extensively tested in more than 25 clinical trials todate and exhibited a relatively benign toxicity profile. In addition toa powerful and specific immunogen the neoantigen peptides may becombined with an adjuvant (e.g., poly-ICLC) or another anti-neoplasticagent. Without being bound by theory, these neoantigens are expected tobypass central thymic tolerance (thus allowing stronger anti-tumor Tcell response), while reducing the potential for autoimmunity (e.g., byavoiding targeting of normal self-antigens). An effective immuneresponse advantageously includes a strong adjuvant to activate theimmune system (Speiser and Romero, Molecularly defined vaccines forcancer immunotherapy, and protective T cell immunity Seminars in Immunol22:144 (2010)). For example, Toll-like receptors (TLRs) have emerged aspowerful sensors of microbial and viral pathogen “danger signals”,effectively inducing the innate immune system, and in turn, the adaptiveimmune system (Bhardwaj and Gnjatic, TLR AGONISTS: Are They GoodAdjuvants? Cancer J. 16:382-391 (2010)). Among the TLR agonists,poly-ICLC (a synthetic double-stranded RNA mimic) is one of the mostpotent activators of myeloid-derived dendritic cells. In a humanvolunteer study, poly-ICLC has been shown to be safe and to induce agene expression profile in peripheral blood cells comparable to thatinduced by one of the most potent live attenuated viral vaccines, theyellow fever vaccine YF-17D (Caskey et al, Synthetic double-stranded RNAinduces innate immune responses similar to a live viral vaccine inhumans J Exp Med 208:2357 (2011)). In a preferred embodiment Hiltonol®,a GMP preparation of poly-ICLC prepared by Oncovir, Inc, is utilized asthe adjuvant. In other embodiments, other adjuvants described herein areenvisioned. For instance oil-in-water, water-in-oil or multiphasicW/O/W; see, e.g., U.S. Pat. No. 7,608,279 and Aucouturier et al, Vaccine19 (2001), 2666-2672, and documents cited therein.

Indications

Examples of cancers and cancer conditions that can be treated with theimmunogenic composition or vaccine of this document include, but are notlimited to a patient in need thereof that has been diagnosed as havingcancer, or at risk of developing cancer. The subject may have a solidtumor such as breast, ovarian, prostate, lung, kidney, gastric, colon,testicular, head and neck, pancreas, brain, melanoma, and other tumorsof tissue organs and hematological tumors, such as lymphomas andleukemias, including acute myelogenous leukemia, chronic myelogenousleukemia, chronic lymphocytic leukemia, T cell lymphocytic leukemia, andB cell lymphomas, tumors of the brain and central nervous system (e.g.,tumors of the meninges, brain, spinal cord, cranial nerves and otherparts of the CNS, such as glioblastomas or medulla blastomas); headand/or neck cancer, breast tumors, tumors of the circulatory system(e.g., heart, mediastinum and pleura, and other intrathoracic organs,vascular tumors, and tumor-associated vascular tissue); tumors of theblood and lymphatic system (e.g., Hodgkin's disease, Non-Hodgkin'sdisease lymphoma, Burkitt's lymphoma, AIDS-related lymphomas, malignantimmunoproliferative diseases, multiple myeloma, and malignant plasmacell neoplasms, lymphoid leukemia, myeloid leukemia, acute or chroniclymphocytic leukemia, monocytic leukemia, other leukemias of specificcell type, leukemia of unspecified cell type, unspecified malignantneoplasms of lymphoid, hematopoietic and related tissues, such asdiffuse large cell lymphoma, T-cell lymphoma or cutaneous T-celllymphoma); tumors of the excretory system (e.g., kidney, renal pelvis,ureter, bladder, and other urinary organs); tumors of thegastrointestinal tract (e.g., esophagus, stomach, small intestine,colon, colorectal, rectosigmoid junction, rectum, anus, and anal canal);tumors involving the liver and intrahepatic bile ducts, gall bladder,and other parts of the biliary tract, pancreas, and other digestiveorgans; tumors of the oral cavity (e.g., lip, tongue, gum, floor ofmouth, palate, parotid gland, salivary glands, tonsil, oropharynx,nasopharynx, puriform sinus, hypopharynx, and other sites of the oralcavity); tumors of the reproductive system (e.g., vulva, vagina, Cervixuteri, uterus, ovary, and other sites associated with female genitalorgans, placenta, penis, prostate, testis, and other sites associatedwith male genital organs); tumors of the respiratory tract (e.g., nasalcavity, middle ear, accessory sinuses, larynx, trachea, bronchus andlung, such as small cell lung cancer and non-small cell lung cancer);tumors of the skeletal system (e.g., bone and articular cartilage oflimbs, bone articular cartilage and other sites); tumors of the skin(e.g., malignant melanoma of the skin, non-melanoma skin cancer, basalcell carcinoma of skin, squamous cell carcinoma of skin, mesothelioma,Kaposi's sarcoma); and tumors involving other tissues includingperipheral nerves and autonomic nervous system, connective and softtissue, retroperitoneoum and peritoneum, eye, thyroid, adrenal gland,and other endocrine glands and related structures, secondary andunspecified malignant neoplasms of lymph nodes, secondary malignantneoplasm of respiratory and digestive systems and secondary malignantneoplasm of other sites.

Of special interest is the treatment of Non-Hodgkin's Lymphoma (NHL),clear cell Renal Cell Carcinoma (ccRCC), metastatic melanoma, sarcoma,leukemia or a cancer of the bladder, colon, brain, breast, head andneck, endometrium, lung, ovary, pancreas or prostate. In certainembodiments, the melanoma is high risk melanoma.

Cancers that can be treated using this immunogenic composition orvaccine may include among others cases which are refractory to treatmentwith other chemotherapeutics. The term “refractory, as used hereinrefers to a cancer (and/or metastases thereof), which shows no or onlyweak antiproliferative response (e.g., no or only weak inhibition oftumor growth) after treatment with another chemotherapeutic agent. Theseare cancers that cannot be treated satisfactorily with otherchemotherapeutics. Refractory cancers encompass not only (i) cancerswhere one or more chemotherapeutics have already failed during treatmentof a patient, but also (ii) cancers that can be shown to be refractoryby other means, e.g., biopsy and culture in the presence ofchemotherapeutics.

The immunogenic composition or vaccine described herein is alsoapplicable to the treatment of patients in need thereof who have notbeen previously treated.

The immunogenic composition or vaccine described herein is alsoapplicable where the subject has no detectable neoplasia but is at highrisk for disease recurrence.

Also of special interest is the treatment of patients in need thereofwho have undergone Autologous Hematopoietic Stem Cell Transplant(AHSCT), and in particular patients who demonstrate residual diseaseafter undergoing AHSCT. The post-AHSCT setting is characterized by a lowvolume of residual disease, the infusion of immune cells to a situationof homeostatic expansion, and the absence of any standardrelapse-delaying therapy. These features provide a unique opportunity touse the described neoplastic vaccine or immunogenic composition to delaydisease relapse.

Pharmaceutical Compositions/Methods of Delivery

The present invention is also directed to pharmaceutical compositionscomprising an effective amount of one or more compounds according to thepresent invention (including a pharmaceutically acceptable salt,thereof), optionally in combination with a pharmaceutically acceptablecarrier, excipient or additive.

While the tumor specific neo-antigenic peptides can be administered asthe sole active pharmaceutical agent, they can also be used incombination with one or more other agents and/or adjuvants. Whenadministered as a combination, the therapeutic agents can be formulatedas separate compositions that are given at the same time or differenttimes, or the therapeutic agents can be given as a single composition.

The compositions may be administered once daily, twice daily, once everytwo days, once every three days, once every four days, once every fivedays, once every six days, once every seven days, once every two weeks,once every three weeks, once every four weeks, once every two months,once every six months, or once per year. The dosing interval can beadjusted according to the needs of individual patients. For longerintervals of administration, extended release or depot formulations canbe used.

The compositions of the invention can be used to treat diseases anddisease conditions that are acute, and may also be used for treatment ofchronic conditions. In particular, the compositions of the invention areused in methods to treat or prevent a neoplasia. In certain embodiments,the compounds of the invention are administered for time periodsexceeding two weeks, three weeks, one month, two months, three months,four months, five months, six months, one year, two years, three years,four years, or five years, ten years, or fifteen years; or for example,any time period range in days, months or years in which the low end ofthe range is any time period between 14 days and 15 years and the upperend of the range is between 15 days and 20 years (e.g., 4 weeks and 15years, 6 months and 20 years). In some cases, it may be advantageous forthe compounds of the invention to be administered for the remainder ofthe patient's life. In preferred embodiments, the patient is monitoredto check the progression of the disease or disorder, and the dose isadjusted accordingly. In preferred embodiments, treatment according tothe invention is effective for at least two weeks, three weeks, onemonth, two months, three months, four months, five months, six months,one year, two years, three years, four years, or five years, ten years,fifteen years, twenty years, or for the remainder of the subject's life.

The tumor specific neo-antigenic peptides may be administered byinjection, orally, parenterally, by inhalation spray, rectally,vaginally, or topically in dosage unit formulations containingconventional pharmaceutically acceptable carriers, adjuvants, andvehicles. The term parenteral as used herein includes, into a lymph nodeor nodes, subcutaneous, intravenous, intramuscular, intrasternal,infusion techniques, intraperitoneally, eye or ocular, intravitreal,intrabuccal, transdermal, intranasal, into the brain, includingintracranial and intradural, into the joints, including ankles, knees,hips, shoulders, elbows, wrists, directly into tumors, and the like, andin suppository form.

Surgical resection uses surgery to remove abnormal tissue in cancer,such as mediastinal, neurogenic, or germ cell tumors, or thymoma. Incertain embodiments, administration of the neoplasia vaccine orimmunogenic composition is initiated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15 or more weeks after tumor resection. Preferably,administration of the neoplasia vaccine or immunogenic composition isinitiated 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks after tumor resection.

Prime/boost regimens refer to the successive administrations of avaccine or immunogenic or immunological compositions. In certainembodiments, administration of the neoplasia vaccine or immunogeniccomposition is in a prime/boost dosing regimen, for exampleadministration of the neoplasia vaccine or immunogenic composition atweeks 1, 2, 3 or 4 as a prime and administration of the neoplasiavaccine or immunogenic composition is at months 2, 3 or 4 as a boost. Inanother embodiment heterologous prime-boost strategies are used toellicit a greater cytotoxic T-cell response (see Schneider et al.,Induction of CD8+ T cells using heterologous prime-boost immunisationstrategies, Immunological Reviews Volume 170, Issue 1, pages 29-38,August 1999). In another embodiment DNA encoding neoantigens is used toprime followed by a protein boost. In another embodiment protein is usedto prime followed by boosting with a virus encoding the neoantigen. Inanother embodiment a virus encoding the neoantigen is used to prime andanother virus is used to boost. In another embodiment protein is used toprime and DNA is used to boost. In a preferred embodiment a DNA vaccineor immunogenic composition is used to prime a T-cell response and arecombinant viral vaccine or immunogenic composition is used to boostthe response. In another preferred embodiment a viral vaccine orimmunogenic composition is coadministered with a protein or DNA vaccineor immunogenic composition to act as an adjuvant for the protein or DNAvaccine or immunogenic composition. The patient can then be boosted witheither the viral vaccine or immunogenic composition, protein, or DNAvaccine or immunogenic composition (see Hutchings et al., Combination ofprotein and viral vaccines induces potent cellular and humoral immuneresponses and enhanced protection from murine malaria challenge. InfectImmun. 2007 December; 75(12):5819-26. Epub 2007 Oct. 1).

The pharmaceutical compositions can be processed in accordance withconventional methods of pharmacy to produce medicinal agents foradministration to patients in need thereof, including humans and othermammals.

Modifications of the neoantigenic peptides can affect the solubility,bioavailability and rate of metabolism of the peptides, thus providingcontrol over the delivery of the active species. Solubility can beassessed by preparing the neoantigenic peptide and testing according toknown methods well within the routine practitioner's skill in the art.

It has been unexpectedly found that a pharmaceutical compositioncomprising succinic acid or a pharmaceutically acceptable salt thereof(succinate) can provide improved solubility for the neoantigenicpeptides. Thus, in one aspect, the invention provides a pharmaceuticalcomposition comprising: at least one neoantigenic peptide or apharmaceutically acceptable salt thereof; a pH modifier (such as a base,such as a dicarboxylate or tricarboxylate salt, for example, apharmaceutically acceptable salt of succinic acid or citric acid); and apharmaceutically acceptable carrier. Such pharmaceutical compositionscan be prepared by combining a solution comprising at least oneneoantigenic peptide with a base, such as a dicarboxylate ortricarboxylate salt, such as a pharmaceutically acceptable salt ofsuccinic acid or citric acid (such as sodium succinate), or by combininga solution comprising at least one neoantigenic peptide with a solutioncomprising a base, such as a dicarboxylate or tricarboxylate salt, suchas a pharmaceutically acceptable salt of succinic acid or citric acid(including, e.g., a succinate buffer solution). In certain embodiments,the pharmaceutical composition comprises sodium succinate. In certainembodiments, the pH modifier (such as citrate or succinate) is presentin the composition at a concentration from about 1 mM to about 10 mM,and, in certain embodiments, at a concentration from about 1.5 mM toabout 7.5 mM, or about 2.0 to about 6.0 mM, or about 3.75 to about 5.0mM.

In certain embodiments of the pharmaceutical composition thepharmaceutically acceptable carrier comprises water. In certainembodiments, the pharmaceutically acceptable carrier further comprisesdextrose. In certain embodiments, the pharmaceutically acceptablecarrier further comprises dimethylsulfoxide. In certain embodiments, thepharmaceutical composition further comprises an immunomodulator oradjuvant. In certain embodiments, the immunodulator or adjuvant isselected from the group consisting of poly-ICLC, 1018 ISS, aluminumsalts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF,IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX,Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312,Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174,OM-197-MP-EC, ONTAK, PEPTEL, vector system, PLGA microparticles,resiquimod, SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, and Aquila's QS21 stimulon. Incertain embodiments, the immunomodulator or adjuvant comprisespoly-ICLC.

Xanthenone derivatives such as, for example, Vadimezan or AsA404 (alsoknown as 5,6-dimethylaxanthenone-4-acetic acid (DMXAA)), may also beused as adjuvants according to embodiments of the invention.Alternatively, such derivatives may also be administered in parallel tothe vaccine or immunogenic composition of the invention, for example viasystemic or intratumoral delivery, to stimulate immunity at the tumorsite. Without being bound by theory, it is believed that such xanthenonederivatives act by stimulating interferon (IFN) production via thestimulator of IFN gene ISTING) receptor (see e.g., Conlon et al. (2013)Mouse, but not Human STING, Binds and Signals in Response to theVascular Disrupting Agent 5,6-Dimethylxanthenone-4-Acetic Acid, Journalof Immunology, 190:5216-25 and Kim et al. (2013) Anticancer Flavonoidsare Mouse-Selective STING Agonists, 8:1396-1401).

The vaccine or immunological composition may also include an adjuvantcompound chosen from the acrylic or methacrylic polymers and thecopolymers of maleic anhydride and an alkenyl derivative. It is inparticular a polymer of acrylic or methacrylic acid cross-linked with apolyalkenyl ether of a sugar or polyalcohol (carbomer), in particularcross-linked with an allyl sucrose or with allylpentaerythritol. It mayalso be a copolymer of maleic anhydride and ethylene cross-linked, forexample, with divinyl ether (see U.S. Pat. No. 6,713,068 herebyincorporated by reference in its entirety).

In certain embodiments, the pH modifier can stabilize the adjuvant orimmunomodulator as described herein.

In certain embodiments, a pharmaceutical composition comprises: one tofive peptides, dimethylsulfoxide (DMSO), dextrose (or trehalose orsucrose), water, succinate, poly I:poly C, poly-L-lysine,carboxymethylcellulose, and chloride. In certain embodiments, each ofthe one to five peptides is present at a concentration of 300 μg/ml. Incertain embodiments, the pharmaceutical composition comprises ≤3% DMSOby volume. In certain embodiments, the pharmaceutical compositioncomprises 3.6-3.7% dextrose in water. In certain embodiments, thepharmaceutical composition comprises 3.6-3.7 mM succinate (e.g., asdisodium succinate) or a salt thereof. In certain embodiments, thepharmaceutical composition comprises 0.5 mg/ml poly I:poly C. In certainembodiments, the pharmaceutical composition comprises 0.375 mg/mlpoly-L-Lysine. In certain embodiments, the pharmaceutical compositioncomprises 1.25 mg/ml sodium carboxymethylcellulose. In certainembodiments, the pharmaceutical composition comprises 0.225% sodiumchloride.

Pharmaceutical compositions comprise the herein-described tumor specificneoantigenic peptides in a therapeutically effective amount for treatingdiseases and conditions (e.g., a neoplasia/tumor), which have beendescribed herein, optionally in combination with a pharmaceuticallyacceptable additive, carrier and/or excipient. One of ordinary skill inthe art from this disclosure and the knowledge in the art will recognizethat a therapeutically effective amount of one of more compoundsaccording to the present invention may vary with the condition to betreated, its severity, the treatment regimen to be employed, thepharmacokinetics of the agent used, as well as the patient (animal orhuman) treated.

To prepare the pharmaceutical compositions according to the presentinvention, a therapeutically effective amount of one or more of thecompounds according to the present invention is preferably intimatelyadmixed with a pharmaceutically acceptable carrier according toconventional pharmaceutical compounding techniques to produce a dose. Acarrier may take a wide variety of forms depending on the form ofpreparation desired for administration, e.g., ocular, oral, topical orparenteral, including gels, creams ointments, lotions and time releasedimplantable preparations, among numerous others. In preparingpharmaceutical compositions in oral dosage form, any of the usualpharmaceutical media may be used. Thus, for liquid oral preparationssuch as suspensions, elixirs and solutions, suitable carriers andadditives including water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents and the like may be used. For solid oralpreparations such as powders, tablets, capsules, and for solidpreparations such as suppositories, suitable carriers and additivesincluding starches, sugar carriers, such as dextrose, mannitol, lactoseand related carriers, diluents, granulating agents, lubricants, binders,disintegrating agents and the like may be used. If desired, the tabletsor capsules may be enteric-coated or sustained release by standardtechniques.

The active compound is included in the pharmaceutically acceptablecarrier or diluent in an amount sufficient to deliver to a patient atherapeutically effective amount for the desired indication, withoutcausing serious toxic effects in the patient treated.

Oral compositions generally include an inert diluent or an ediblecarrier. They may be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound or its prodrug derivative can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Pharmaceuticallycompatible binding agents, and/or adjuvant materials can be included aspart of the composition.

The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a dispersing agent such as alginicacid or corn starch; a lubricant such as magnesium stearate; a glidantsuch as colloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, methyl salicylate,or orange flavoring. When the dosage unit form is a capsule, it cancontain, in addition to material herein discussed, a liquid carrier suchas a fatty oil. In addition, dosage unit forms can contain various othermaterials which modify the physical form of the dosage unit, forexample, coatings of sugar, shellac, or enteric agents.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil emulsion and as a bolus, etc.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface-active ordispersing agent. Molded tablets may be made by molding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets optionally may be coated or scored and maybe formulated so as to provide slow or controlled release of the activeingredient therein.

Methods of formulating such slow or controlled release compositions ofpharmaceutically active ingredients, are known in the art and describedin several issued US patents, some of which include, but are not limitedto, U.S. Pat. Nos. 3,870,790; 4,226,859; 4,369,172; 4,842,866 and5,705,190, the disclosures of which are incorporated herein by referencein their entireties. Coatings can be used for delivery of compounds tothe intestine (see, e.g., U.S. Pat. Nos. 6,638,534, 5,541,171,5,217,720, and 6,569,457, and references cited therein).

The active compound or pharmaceutically acceptable salt thereof may alsobe administered as a component of an elixir, suspension, syrup, wafer,chewing gum or the like. A syrup may contain, in addition to the activecompounds, sucrose or fructose as a sweetening agent and certainpreservatives, dyes and colorings and flavors.

Solutions or suspensions used for ocular, parenteral, intradermal,subcutaneous, or topical application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates; and agents for theadjustment of tonicity such as sodium chloride or dextrose.

In certain embodiments, the pharmaceutically acceptable carrier is anaqueous solvent, i.e., a solvent comprising water, optionally withadditional co-solvents. Exemplary pharmaceutically acceptable carriersinclude water, buffer solutions in water (such as phosphate-bufferedsaline (PBS), and 5% dextrose in water (D5W) or 10% trehalose or 10%sucrose. In certain embodiments, the aqueous solvent further comprisesdimethyl sulfoxide (DMSO), e.g., in an amount of about 1-4%, or 1-3%. Incertain embodiments, the pharmaceutically acceptable carrier is isotonic(i.e., has substantially the same osmotic pressure as a body fluid suchas plasma).

In one embodiment, the active compounds are prepared with carriers thatprotect the compound against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, polylactic acid, and polylactic-co-glycolicacid (PLGA). Methods for preparation of such formulations are within theambit of the skilled artisan in view of this disclosure and theknowledge in the art.

A skilled artisan from this disclosure and the knowledge in the artrecognizes that in addition to tablets, other dosage forms can beformulated to provide slow or controlled release of the activeingredient. Such dosage forms include, but are not limited to, capsules,granulations and gel-caps.

Liposomal suspensions may also be pharmaceutically acceptable carriers.These may be prepared according to methods known to those skilled in theart. For example, liposomal formulations may be prepared by dissolvingappropriate lipid(s) in an inorganic solvent that is then evaporated,leaving behind a thin film of dried lipid on the surface of thecontainer. An aqueous solution of the active compound are thenintroduced into the container. The container is then swirled by hand tofree lipid material from the sides of the container and to disperselipid aggregates, thereby forming the liposomal suspension. Othermethods of preparation well known by those of ordinary skill may also beused in this aspect of the present invention.

The formulations may conveniently be presented in unit dosage form andmay be prepared by conventional pharmaceutical techniques. Suchtechniques include the step of bringing into association the activeingredient and the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredient with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

Formulations and compositions suitable for topical administration in themouth include lozenges comprising the ingredients in a flavored basis,usually sucrose and acacia or tragacanth; pastilles comprising theactive ingredient in an inert basis such as gelatin and glycerin, orsucrose and acacia; and mouthwashes comprising the ingredient to beadministered in a suitable liquid carrier.

Formulations suitable for topical administration to the skin may bepresented as ointments, creams, gels and pastes comprising theingredient to be administered in a pharmaceutical acceptable carrier. Apreferred topical delivery system is a transdermal patch containing theingredient to be administered.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate.

Formulations suitable for nasal administration, wherein the carrier is asolid, include a coarse powder having a particle size, for example, inthe range of 20 to 500 microns which is administered in the manner inwhich snuff is administered, i.e., by rapid inhalation through the nasalpassage from a container of the powder held close up to the nose.Suitable formulations, wherein the carrier is a liquid, foradministration, as for example, a nasal spray or as nasal drops, includeaqueous or oily solutions of the active ingredient.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

The parenteral preparation can be enclosed in ampoules, disposablesyringes or multiple dose vials made of glass or plastic. Ifadministered intravenously, preferred carriers include, for example,physiological saline or phosphate buffered saline (PBS).

For parenteral formulations, the carrier usually comprises sterile wateror aqueous sodium chloride solution, though other ingredients includingthose which aid dispersion may be included. Of course, where sterilewater is to be used and maintained as sterile, the compositions andcarriers are also sterilized. Injectable suspensions may also beprepared, in which case appropriate liquid carriers, suspending agentsand the like may be employed.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain antioxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example, water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Administration of the active compound may range from continuous(intravenous drip) to several oral administrations per day (for example,Q.I.D.) and may include oral, topical, eye or ocular, parenteral,intramuscular, intravenous, sub-cutaneous, transdermal (which mayinclude a penetration enhancement agent), buccal and suppositoryadministration, among other routes of administration, including throughan eye or ocular route.

The neoplasia vaccine or immunogenic composition may be administered byinjection, orally, parenterally, by inhalation spray, rectally,vaginally, or topically in dosage unit formulations containingconventional pharmaceutically acceptable carriers, adjuvants, andvehicles. The term parenteral as used herein includes, into a lymph nodeor nodes, subcutaneous, intravenous, intramuscular, intrasternal,infusion techniques, intraperitoneally, eye or ocular, intravitreal,intrabuccal, transdermal, intranasal, into the brain, includingintracranial and intradural, into the joints, including ankles, knees,hips, shoulders, elbows, wrists, directly into tumors, and the like, andin suppository form.

Various techniques can be used for providing the subject compositions atthe site of interest, such as injection, use of catheters, trocars,projectiles, pluronic gel, stents, sustained drug release polymers orother device which provides for internal access. Where an organ ortissue is accessible because of removal from the patient, such organ ortissue may be bathed in a medium containing the subject compositions,the subject compositions may be painted onto the organ, or may beapplied in any convenient way.

The tumor specific neoantigenic peptides may be administered through adevice suitable for the controlled and sustained release of acomposition effective in obtaining a desired local or systemicphysiological or pharmacological effect. The method includes positioningthe sustained released drug delivery system at an area wherein releaseof the agent is desired and allowing the agent to pass through thedevice to the desired area of treatment.

The tumor specific neoantigenic peptides may be utilized in combinationwith at least one known other therapeutic agent, or a pharmaceuticallyacceptable salt of said agent. Examples of known therapeutic agentswhich can be used include, but are not limited to, corticosteroids(e.g., cortisone, prednisone, dexamethasone), non-steroidalanti-inflammatory drugs (NSAIDS) (e.g., ibuprofen, celecoxib, aspirin,indomethicin, naproxen), alkylating agents such as busulfan, cis-platin,mitomycin C, and carboplatin; antimitotic agents such as colchicine,vinblastine, paclitaxel, and docetaxel; topo I inhibitors such ascamptothecin and topotecan; topo II inhibitors such as doxorubicin andetoposide; and/or RNA/DNA antimetabolites such as 5-azacytidine,5-fluorouracil and methotrexate; DNA antimetabolites such as5-fluoro-2′-deoxy-uridine, ara-C, hydroxyurea and thioguanine;antibodies such as HERCEPTIN and RITUXAN.

It should be understood that in addition to the ingredients particularlymentioned herein, the formulations of the present invention may includeother agents conventional in the art having regard to the type offormulation in question, for example, those suitable for oraladministration may include flavoring agents.

Pharmaceutically acceptable salt forms may be the preferred chemicalform of compounds according to the present invention for inclusion inpharmaceutical compositions according to the present invention.

The present compounds or their derivatives, including prodrug forms ofthese agents, can be provided in the form of pharmaceutically acceptablesalts. As used herein, the term pharmaceutically acceptable salts orcomplexes refers to appropriate salts or complexes of the activecompounds according to the present invention which retain the desiredbiological activity of the parent compound and exhibit limitedtoxicological effects to normal cells. Nonlimiting examples of suchsalts are (a) acid addition salts formed with inorganic acids (forexample, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoricacid, nitric acid, and the like), and salts formed with organic acidssuch as acetic acid, oxalic acid, tartaric acid, succinic acid, malicacid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginicacid, and polyglutamic acid, among others; (b) base addition saltsformed with metal cations such as zinc, calcium, sodium, potassium, andthe like, among numerous others.

The compounds herein are commercially available or can be synthesized.As can be appreciated by the skilled artisan, further methods ofsynthesizing the compounds of the formulae herein is evident to those ofordinary skill in the art. Additionally, the various synthetic steps maybe performed in an alternate sequence or order to give the desiredcompounds. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, 2nd. Ed., Wiley-VCH Publishers (1999); T. W. Greene andP. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd. Ed., JohnWiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser'sReagents for Organic Synthesis, John Wiley and Sons (1999); and L.Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, JohnWiley and Sons (1995), and subsequent editions thereof.

The additional agents that may be included with the tumor specificneo-antigenic peptides of this invention may contain one or moreasymmetric centers and thus occur as racemates and racemic mixtures,single enantiomers, individual diastereomers and diastereomericmixtures. All such isomeric forms of these compounds are expresslyincluded in the present invention. The compounds of this invention mayalso be represented in multiple tautomeric forms, in such instances, theinvention expressly includes all tautomeric forms of the compoundsdescribed herein (e.g., alkylation of a ring system may result inalkylation at multiple sites, the invention expressly includes all suchreaction products). All such isomeric forms of such compounds areexpressly included in the present invention. All crystal forms of thecompounds described herein are expressly included in the presentinvention.

Dosage

When the agents described herein are administered as pharmaceuticals tohumans or animals, they can be given per se or as a pharmaceuticalcomposition containing active ingredient in combination with apharmaceutically acceptable carrier, excipient, or diluent.

Actual dosage levels and time course of administration of the activeingredients in the pharmaceutical compositions of the invention can bevaried so as to obtain an amount of the active ingredient which iseffective to achieve the desired therapeutic response for a particularpatient, composition, and mode of administration, without being toxic tothe patient. Generally, agents or pharmaceutical compositions of theinvention are administered in an amount sufficient to reduce oreliminate symptoms associated with viral infection and/or autoimmunedisease.

A preferred dose of an agent is the maximum that a patient can tolerateand not develop serious or unacceptable side effects. Exemplary doseranges include 0.01 mg to 250 mg per day, 0.01 mg to 100 mg per day, 1mg to 100 mg per day, 10 mg to 100 mg per day, 1 mg to 10 mg per day,and 0.01 mg to 10 mg per day. A preferred dose of an agent is themaximum that a patient can tolerate and not develop serious orunacceptable side effects. In embodiments, the agent is administered ata concentration of about 10 micrograms to about 100 mg per kilogram ofbody weight per day, about 0.1 to about 10 mg/kg per day, or about 1.0mg to about 10 mg/kg of body weight per day.

In embodiments, the pharmaceutical composition comprises an agent in anamount ranging between 1 and 10 mg, such as 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 mg.

In embodiments, the therapeutically effective dosage produces a serumconcentration of an agent of from about 0.1 ng/ml to about 50-100 mglml.The pharmaceutical compositions 5 typically should provide a dosage offrom about 0.001 mg to about 2000 mg of compound per kilogram of bodyweight per day. For example, dosages for systemic administration to ahuman patient can range from 1-10 mglkg, 20-80 mglkg, 5-50 mg/kg, 75-150mg/kg, 100-500 mglkg, 250-750 mglkg, 500-1000 mglkg, 1-10 mg/kg, 5-50mg/kg, 25-75 mg/kg, 50-100 mg/kg, 100-250 mg/kg, 50-100 mg/kg, 250-500mg/kg, 500-750 mg/kg, 750-1000 mg/kg, 1000-1500 mg/kg, 10 1500-2000mg/kg, 5 mg/kg, 20 mg/kg, 50 mg/kg, 100 mg/kg, 500 mg/kg, 1000 mg/kg,1500 mg/kg, or 2000 mg/kg. Pharmaceutical dosage unit forms are preparedto provide from about 1 mg to about 5000 mg, for example from about 100to about 2500 mg of the compound or a combination of essentialingredients per dosage unit form.

In embodiments, about 50 nM to about 1 μM of an agent is administered toa subject. In related embodiments, about 50-100 nM, 50-250 nM, 100-500nM, 250-500 nM, 250-750 nM, 500-750 nM, 500 nM to 1 μM, or 750 nM to 1μM of an agent is administered to a subject.

Determination of an effective amount is well within the capability ofthose skilled in the art, especially in light of the detailed disclosureprovided herein. Generally, an efficacious or effective amount of anagent is determined by first administering a low dose of the agent(s)and then incrementally increasing the administered dose or dosages untila desired effect (e.g., reduce or eliminate symptoms associated withviral infection or autoimmune disease) is observed in the treatedsubject, with minimal or acceptable toxic side effects. Applicablemethods for determining an appropriate dose and dosing schedule foradministration of a pharmaceutical composition of the present inventionare described, for example, in Goodman and Gilman's The PharmacologicalBasis of Therapeutics, Goodman et al., eds., 11th Edition, McGraw-Hill2005, and Remington: The Science and Practice of Pharmacy, 20th and 21stEditions, Gennaro and University of the Sciences in Philadelphia, Eds.,Lippencott Williams & Wilkins (2003 and 2005), each of which is herebyincorporated by reference.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, as herein discussed, or an appropriate fractionthereof, of the administered ingredient.

The dosage regimen for treating a disorder or a disease with the tumorspecific neoantigenic peptides of this invention and/or compositions ofthis invention is based on a variety of factors, including the type ofdisease, the age, weight, sex, medical condition of the patient, theseverity of the condition, the route of administration, and theparticular compound employed. Thus, the dosage regimen may vary widely,but can be determined routinely using standard methods.

The amounts and dosage regimens administered to a subject can depend ona number of factors, such as the mode of administration, the nature ofthe condition being treated, the body weight of the subject beingtreated and the judgment of the prescribing physician; all such factorsbeing within the ambit of the skilled artisan from this disclosure andthe knowledge in the art.

The amount of compound included within therapeutically activeformulations according to the present invention is an effective amountfor treating the disease or condition. In general, a therapeuticallyeffective amount of the present preferred compound in dosage formusually ranges from slightly less than about 0.025 mg/kg/day to about2.5 g/kg/day, preferably about 0.1 mg/kg/day to about 100 mg/kg/day ofthe patient or considerably more, depending upon the compound used, thecondition or infection treated and the route of administration, althoughexceptions to this dosage range may be contemplated by the presentinvention. In its most preferred form, compounds according to thepresent invention are administered in amounts ranging from about 1mg/kg/day to about 100 mg/kg/day. The dosage of the compound can dependon the condition being treated, the particular compound, and otherclinical factors such as weight and condition of the patient and theroute of administration of the compound. It is to be understood that thepresent invention has application for both human and veterinary use.

For oral administration to humans, a dosage of between approximately 0.1to 100 mg/kg/day, preferably between approximately 1 and 100 mg/kg/day,is generally sufficient.

Where drug delivery is systemic rather than topical, this dosage rangegenerally produces effective blood level concentrations of activecompound ranging from less than about 0.04 to about 400 micrograms/cc ormore of blood in the patient. The compound is conveniently administeredin any suitable unit dosage form, including but not limited to onecontaining 0.001 to 3000 mg, preferably 0.05 to 500 mg of activeingredient per unit dosage form. An oral dosage of 10-250 mg is usuallyconvenient.

According to certain exemplary embodiments, the vaccine or immunogeniccomposition is administered at a dose of about 10 μg-1 mg perneoantigenic peptide. According to certain exemplary embodiments, thevaccine or immunogenic composition is administered at an average weeklydose level of about 10 μg-2000 μg per neoantigenic peptide.

The concentration of active compound in the drug composition will dependon absorption, distribution, inactivation, and excretion rates of thedrug as well as other factors known to those of skill in the art. It isto be noted that dosage values will also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed composition. The active ingredient may be administered atonce, or may be divided into a number of smaller doses to beadministered at varying intervals of time.

The invention provides for pharmaceutical compositions containing atleast one tumor specific neoantigen described herein. In embodiments,the pharmaceutical compositions contain a pharmaceutically acceptablecarrier, excipient, or diluent, which includes any pharmaceutical agentthat does not itself induce the production of an immune response harmfulto a subject receiving the composition, and which may be administeredwithout undue toxicity. As used herein, the term “pharmaceuticallyacceptable” means being approved by a regulatory agency of the Federalor a state government or listed in the U.S. Pharmacopia, EuropeanPharmacopia or other generally recognized pharmacopia for use inmammals, and more particularly in humans. These compositions can beuseful for treating and/or preventing viral infection and/or autoimmunedisease.

A thorough discussion of pharmaceutically acceptable carriers, diluents,and other excipients is presented in Remington's Pharmaceutical Sciences(17th ed., Mack Publishing Company) and Remington: The Science andPractice of Pharmacy (21st ed., Lippincott Williams & Wilkins), whichare hereby incorporated by reference. The formulation of thepharmaceutical composition should suit the mode of administration. Inembodiments, the pharmaceutical composition is suitable foradministration to humans, and can be sterile, non-particulate and/ornon-pyrogenic.

Pharmaceutically acceptable carriers, excipients, or diluents include,but are not limited, to saline, buffered saline, dextrose, water,glycerol, ethanol, sterile isotonic aqueous buffer, and combinationsthereof.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives, and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include, but arenot limited to: (1) water soluble antioxidants, such as ascorbic acid,cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodiumsulfite and the like; (2) oil-soluble antioxidants, such as ascorbylpalmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene(BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3)metal chelating agents, such as citric acid, ethylenediamine tetraaceticacid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

In embodiments, the pharmaceutical composition is provided in a solidform, such as a lyophilized powder suitable for reconstitution, a liquidsolution, suspension, emulsion, tablet, pill, capsule, sustained releaseformulation, or powder.

In embodiments, the pharmaceutical composition is supplied in liquidform, for example, in a sealed container indicating the quantity andconcentration of the active ingredient in the pharmaceuticalcomposition. In related embodiments, the liquid form of thepharmaceutical composition is supplied in a hermetically sealedcontainer.

Methods for formulating the pharmaceutical compositions of the presentinvention are conventional and well known in the art (see Remington andRemington's). One of skill in the art can readily formulate apharmaceutical composition having the desired characteristics (e.g.,route of administration, biosafety, and release profile).

Methods for preparing the pharmaceutical compositions include the stepof bringing into association the active ingredient with apharmaceutically acceptable carrier and, optionally, one or moreaccessory ingredients. The pharmaceutical compositions can be preparedby uniformly and intimately bringing into association the activeingredient with liquid carriers, or finely divided solid carriers, orboth, and then, if necessary, shaping the product. Additionalmethodology for preparing the pharmaceutical compositions, including thepreparation of multilayer dosage forms, are described in Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems (9th ed.,Lippincott Williams & Wilkins), which is hereby incorporated byreference.

Pharmaceutical compositions suitable for oral administration can be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound(s) describedherein, a derivative thereof, or a pharmaceutically acceptable salt orprodrug thereof as the active ingredient(s). The active ingredient canalso be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (e.g., capsules, tablets,pills, dragees, powders, granules and the like), the active ingredientis mixed with one or more pharmaceutically acceptable carriers,excipients, or diluents, such as sodium citrate or dicalcium phosphate,and/or any of the following: (1) fillers or extenders, such as starches,lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders,such as, for example, carboxymethylcellulose, alginates, gelatin,polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such asglycerol; (4) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and sodium carbonate; (5) solution retarding agents, such as paraffin;(6) absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, acetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets, and pills, thepharmaceutical compositions can also comprise buffering agents. Solidcompositions of a similar type can also be prepared using fillers insoft and hard-filled gelatin capsules, and excipients such as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet can be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets can be prepared usingbinders (for example, gelatin or hydroxypropylmethyl cellulose),lubricants, inert diluents, preservatives, disintegrants (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-actives, and/or dispersing agents. Molded tablets can be made bymolding in a suitable machine a mixture of the powdered activeingredient moistened with an inert liquid diluent.

The tablets and other solid dosage forms, such as dragees, capsules,pills, and granules, can optionally be scored or prepared with coatingsand shells, such as enteric coatings and other coatings well known inthe art.

In some embodiments, in order to prolong the effect of an activeingredient, it is desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This can be accomplished by theuse of a liquid suspension of crystalline or amorphous material havingpoor water solubility. The rate of absorption of the active ingredientthen depends upon its rate of dissolution which, in turn, can dependupon crystal size and crystalline form. Alternatively, delayedabsorption of a parenterally-administered active ingredient isaccomplished by dissolving or suspending the compound in an oil vehicle.In addition, prolonged absorption of the injectable pharmaceutical formcan be brought about by the inclusion of agents that delay absorptionsuch as aluminum monostearate and gelatin.

Controlled release parenteral compositions can be in form of aqueoussuspensions, microspheres, microcapsules, magnetic microspheres, oilsolutions, oil suspensions, emulsions, or the active ingredient can beincorporated in biocompatible carrier(s), liposomes, nanoparticles,implants or infusion devices.

Materials for use in the preparation of microspheres and/ormicrocapsules include biodegradable/bioerodible polymers such aspolyglactin, poly-(isobutyl cyanoacrylate),poly(2-hydroxyethyl-L-glutamine) and poly(lactic acid).

Biocompatible carriers which can be used when formulating a controlledrelease parenteral formulation include carbohydrates such as dextrans,proteins such as albumin, lipoproteins or antibodies.

Materials for use in implants can be non-biodegradable, e.g.,polydimethylsiloxane, or biodegradable such as, e.g.,poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(orthoesters).

In embodiments, the active ingredient(s) are administered by aerosol.This is accomplished by preparing an aqueous aerosol, liposomalpreparation, or solid particles containing the compound. A nonaqueous(e.g., fluorocarbon propellant) suspension can be used. Thepharmaceutical composition can also be administered using a sonicnebulizer, which would minimize exposing the agent to shear, which canresult in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the active ingredient(s) together withconventional pharmaceutically-acceptable carriers and stabilizers. Thecarriers and stabilizers vary with the requirements of the particularcompound, but typically include nonionic surfactants (Tweens, Pluronics,or polyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions.

Dosage forms for topical or transdermal administration of an activeingredient(s) includes powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The activeingredient(s) can be mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants as appropriate.

Transdermal patches suitable for use in the present invention aredisclosed in Transdermal Drug Delivery: Developmental Issues andResearch Initiatives (Marcel Dekker Inc., 1989) and U.S. Pat. Nos.4,743,249, 4,906,169, 5,198,223, 4,816,540, 5,422,119, 5,023,084, whichare hereby incorporated by reference. The transdermal patch can also beany transdermal patch well known in the art, including transscrotalpatches. Pharmaceutical compositions in such transdermal patches cancontain one or more absorption enhancers or skin permeation enhancerswell known in the art (see, e.g., U.S. Pat. Nos. 4,379,454 and4,973,468, which are hereby incorporated by reference). Transdermaltherapeutic systems for use in the present invention can be based oniontophoresis, diffusion, or a combination of these two effects.

Transdermal patches have the added advantage of providing controlleddelivery of active ingredient(s) to the body. Such dosage forms can bemade by dissolving or dispersing the active ingredient(s) in a propermedium. Absorption enhancers can also be used to increase the flux ofthe active ingredient across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe active ingredient(s) in a polymer matrix or gel.

Such pharmaceutical compositions can be in the form of creams,ointments, lotions, liniments, gels, hydrogels, solutions, suspensions,sticks, sprays, pastes, plasters and other kinds of transdermal drugdelivery systems. The compositions can also include pharmaceuticallyacceptable carriers or excipients such as emulsifying agents,antioxidants, buffering agents, preservatives, humectants, penetrationenhancers, chelating agents, gel-forming agents, ointment bases,perfumes, and skin protective agents.

Examples of emulsifying agents include, but are not limited to,naturally occurring gums, e.g. gum acacia or gum tragacanth, naturallyoccurring phosphatides, e.g. soybean lecithin and sorbitan monooleatederivatives.

Examples of antioxidants include, but are not limited to, butylatedhydroxy anisole (BHA), ascorbic acid and derivatives thereof, tocopheroland derivatives thereof, and cysteine.

Examples of preservatives include, but are not limited to, parabens,such as methyl or propyl p-hydroxybenzoate and benzalkonium chloride.

Examples of humectants include, but are not limited to, glycerin,propylene glycol, sorbitol and urea.

Examples of penetration enhancers include, but are not limited to,propylene glycol, DMSO, triethanolamine, N,N-dimethylacetamide,N,N-dimethylformamide, 2-pyrrolidone and derivatives thereof,tetrahydrofurfuryl alcohol, propylene glycol, diethylene glycolmonoethyl or monomethyl ether with propylene glycol monolaurate ormethyl laurate, eucalyptol, lecithin, TRANSCUTOL, and AZONE.

Examples of chelating agents include, but are not limited to, sodiumEDTA, citric acid and phosphoric acid.

Examples of gel forming agents include, but are not limited to,Carbopol, cellulose derivatives, bentonite, alginates, gelatin andpolyvinylpyrrolidone.

In addition to the active ingredient(s), the ointments, pastes, creams,and gels of the present invention can contain excipients, such as animaland vegetable fats, oils, waxes, paraffins, starch, tragacanth,cellulose derivatives, polyethylene glycols, silicones, bentonites,silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain excipients such as lactose, talc, silicicacid, aluminum hydroxide, calcium silicates and polyamide powder, ormixtures of these substances. Sprays can additionally contain customarypropellants, such as chlorofluorohydrocarbons, and volatileunsubstituted hydrocarbons, such as butane and propane.

Injectable depot forms are made by forming microencapsule matrices ofcompound(s) of the invention in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of compound topolymer, and the nature of the particular polymer employed, the rate ofcompound release can be controlled. Examples of other biodegradablepolymers include poly(orthoesters) and poly(anhydrides). Depotinjectable formulations are also prepared by entrapping the drug inliposomes or microemulsions which are compatible with body tissue.

Subcutaneous implants are well known in the art and are suitable for usein the present invention. Subcutaneous implantation methods arepreferably non-irritating and mechanically resilient. The implants canbe of matrix type, of reservoir type, or hybrids thereof. In matrix typedevices, the carrier material can be porous or non-porous, solid orsemi-solid, and permeable or impermeable to the active compound orcompounds. The carrier material can be biodegradable or may slowly erodeafter administration. In some instances, the matrix is non-degradablebut instead relies on the diffusion of the active compound through thematrix for the carrier material to degrade. Alternative subcutaneousimplant methods utilize reservoir devices where the active compound orcompounds are surrounded by a rate controlling membrane, e.g., amembrane independent of component concentration (possessing zero-orderkinetics). Devices consisting of a matrix surrounded by a ratecontrolling membrane also suitable for use.

Both reservoir and matrix type devices can contain materials such aspolydimethylsiloxane, such as SILASTIC, or other silicone rubbers.Matrix materials can be insoluble polypropylene, polyethylene, polyvinylchloride, ethylvinyl acetate, polystyrene and polymethacrylate, as wellas glycerol esters of the glycerol palmitostearate, glycerol stearate,and glycerol behenate type. Materials can be hydrophobic or hydrophilicpolymers and optionally contain solubilizing agents.

Subcutaneous implant devices can be slow-release capsules made with anysuitable polymer, e.g., as described in U.S. Pat. Nos. 5,035,891 and4,210,644, which are hereby incorporated by reference.

In general, at least four different approaches are applicable in orderto provide rate control over the release and transdermal permeation of adrug compound. These approaches are: membrane-moderated systems,adhesive diffusion-controlled systems, matrix dispersion-type systemsand microreservoir systems. It is appreciated that a controlled releasepercutaneous and/or topical composition can be obtained by using asuitable mixture of these approaches.

In a membrane-moderated system, the active ingredient is present in areservoir which is totally encapsulated in a shallow compartment moldedfrom a drug-impermeable laminate, such as a metallic plastic laminate,and a rate-controlling polymeric membrane such as a microporous or anon-porous polymeric membrane, e.g., ethylene-vinyl acetate copolymer.The active ingredient is released through the rate controlling polymericmembrane. In the drug reservoir, the active ingredient can either bedispersed in a solid polymer matrix or suspended in an unleachable,viscous liquid medium such as silicone fluid. On the external surface ofthe polymeric membrane, a thin layer of an adhesive polymer is appliedto achieve an intimate contact of the transdermal system with the skinsurface. The adhesive polymer is preferably a polymer which ishypoallergenic and compatible with the active drug substance.

In an adhesive diffusion-controlled system, a reservoir of the activeingredient is formed by directly dispersing the active ingredient in anadhesive polymer and then by, e.g., solvent casting, spreading theadhesive containing the active ingredient onto a flat sheet ofsubstantially drug-impermeable metallic plastic backing to form a thindrug reservoir layer.

A matrix dispersion-type system is characterized in that a reservoir ofthe active ingredient is formed by substantially homogeneouslydispersing the active ingredient in a hydrophilic or lipophilic polymermatrix. The drug-containing polymer is then molded into disc with asubstantially well-defined surface area and controlled thickness. Theadhesive polymer is spread along the circumference to form a strip ofadhesive around the disc.

A microreservoir system can be considered as a combination of thereservoir and matrix dispersion type systems. In this case, thereservoir of the active substance is formed by first suspending the drugsolids in an aqueous solution of water-soluble polymer and thendispersing the drug suspension in a lipophilic polymer to form amultiplicity of unleachable, microscopic spheres of drug reservoirs.

Any of the herein-described controlled release, extended release, andsustained release compositions can be formulated to release the activeingredient in about 30 minutes to about 1 week, in about 30 minutes toabout 72 hours, in about 30 minutes to 24 hours, in about 30 minutes to12 hours, in about 30 minutes to 6 hours, in about 30 minutes to 4hours, and in about 3 hours to 10 hours. In embodiments, an effectiveconcentration of the active ingredient(s) is sustained in a subject for4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 24 hours, 48hours, 72 hours, or more after administration of the pharmaceuticalcompositions to the subject.

Vaccine or Immunogenic Compositions

The present invention is directed to an immunogenic composition, e.g., aneoplasia vaccine or immunogenic composition capable of raising aspecific T-cell response. The neoplasia vaccine or immunogeniccomposition comprises neoantigenic peptides and/or neoantigenicpolypeptides corresponding to tumor specific neoantigens identified bythe methods described herein.

A suitable neoplasia vaccine or immunogenic composition can preferablycontain a plurality of tumor specific neoantigenic peptides. In anembodiment, the vaccine or immunogenic composition can include between 1and 100 sets of peptides, more preferably between 1 and 50 suchpeptides, even more preferably between 10 and 30 sets peptides, evenmore preferably between 15 and 25 peptides. According to anotherpreferred embodiment, the vaccine or immunogenic composition can includeat least one peptides, more preferably 2, 3, 4, or 5 peptides, Incertain embodiments, the vaccine or immunogenic composition can comprise5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 different peptides.

The optimum amount of each peptide to be included in the vaccine orimmunogenic composition and the optimum dosing regimen can be determinedby one skilled in the art without undue experimentation. For example,the peptide or its variant may be prepared for intravenous (i.v.)injection, sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c, i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c,i.p. and i.v. For example, doses of between 1 and 500 mg 50 μg and 1.5mg, preferably 10 μg to 500 μg, of peptide or DNA may be given and candepend from the respective peptide or DNA. Doses of this range weresuccessfully used in previous trials (Brunsvig P F, et al., CancerImmunol Immunother. 2006; 55(12): 1553-1564; M. Staehler, et al., ASCOmeeting 2007; Abstract No 3017). Other methods of administration of thevaccine or immunogenic composition are known to those skilled in theart.

In one embodiment of the present invention the different tumor specificneoantigenic peptides and/or polypeptides are selected for use in theneoplasia vaccine or immunogenic composition so as to maximize thelikelihood of generating an immune attack against the neoplasia/tumor ofthe patient. Without being bound by theory, it is believed that theinclusion of a diversity of tumor specific neoantigenic peptides cangenerate a broad scale immune attack against a neoplasia/tumor. In oneembodiment, the selected tumor specific neoantigenicpeptides/polypeptides are encoded by missense mutations. In a secondembodiment, the selected tumor specific neoantigenicpeptides/polypeptides are encoded by a combination of missense mutationsand neoORF mutations. In a third embodiment, the selected tumor specificneoantigenic peptides/polypeptides are encoded by neoORF mutations.

In one embodiment in which the selected tumor specific neoantigenicpeptides/polypeptides are encoded by missense mutations, the peptidesand/or polypeptides are chosen based on their capability to associatewith the particular MHC molecules of the patient. Peptides/polypeptidesderived from neoORF mutations can also be selected on the basis of theircapability to associate with the particular MHC molecules of thepatient, but can also be selected even if not predicted to associatewith the particular MHC molecules of the patient.

The vaccine or immunogenic composition is capable of raising a specificcytotoxic T-cells response and/or a specific helper T-cell response.

The vaccine or immunogenic composition can further comprise an adjuvantand/or a carrier. Examples of useful adjuvants and carriers are givenherein herein. The peptides and/or polypeptides in the composition canbe associated with a carrier such as, e.g., a protein or anantigen-presenting cell such as e.g. a dendritic cell (DC) capable ofpresenting the peptide to a T-cell.

Adjuvants are any substance whose admixture into the vaccine orimmunogenic composition increases or otherwise modifies the immuneresponse to the mutant peptide. Carriers are scaffold structures, forexample a polypeptide or a polysaccharide, to which the neoantigenicpeptides, is capable of being associated. Optionally, adjuvants areconjugated covalently or non-covalently to the peptides or polypeptidesof the invention.

The ability of an adjuvant to increase the immune response to an antigenis typically manifested by a significant increase in immune-mediatedreaction, or reduction in disease symptoms. For example, an increase inhumoral immunity is typically manifested by a significant increase inthe titer of antibodies raised to the antigen, and an increase in T-cellactivity is typically manifested in increased cell proliferation, orcellular cytotoxicity, or cytokine secretion. An adjuvant may also alteran immune response, for example, by changing a primarily humoral or Th2response into a primarily cellular, or Th1 response.

Suitable adjuvants include, but are not limited to 1018 ISS, aluminumsalts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF,IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX,Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312,Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174,OM-197-MP-EC, ONTAK, PEPTEL. vector system, PLG microparticles,resiquimod, SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon (AquilaBiotech, Worcester, Mass., USA) which is derived from saponin,mycobacterial extracts and synthetic bacterial cell wall mimics, andother proprietary adjuvants such as Ribi's Detox. Quil or Superfos.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Dupuis M, etal., Cell Immunol. 1998; 186(1): 18-27; Allison A C; Dev Biol Stand.1998; 92:3-11). Also cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-alpha), accelerating the maturation of dendriticcells into efficient antigen-presenting cells for T-lymphocytes (e.g.,GM-CSF, IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specificallyincorporated herein by reference in its entirety) and acting asimmunoadjuvants (e.g., IL-12) (Gabrilovich D I, et al., J ImmunotherEmphasis Tumor Immunol. 1996 (6):414-418).

Toll like receptors (TLRs) may also be used as adjuvants, and areimportant members of the family of pattern recognition receptors (PRRs)which recognize conserved motifs shared by many micro-organisms, termed“pathogen-associated molecular patterns” (PAMPS). Recognition of these“danger signals” activates multiple elements of the innate and adaptiveimmune system. TLRs are expressed by cells of the innate and adaptiveimmune systems such as dendritic cells (DCs), macrophages, T and Bcells, mast cells, and granulocytes and are localized in differentcellular compartments, such as the plasma membrane, lysosomes,endosomes, and endolysosomes. Different TLRs recognize distinct PAMPS.For example, TLR4 is activated by LPS contained in bacterial cell walls,TLR9 is activated by unmethylated bacterial or viral CpG DNA, and TLR3is activated by double stranded RNA. TLR ligand binding leads to theactivation of one or more intracellular signaling pathways, ultimatelyresulting in the production of many key molecules associated withinflammation and immunity (particularly the transcription factor NF-κBand the Type-I interferons). TLR mediated DC activation leads toenhanced DC activation, phagocytosis, upregulation of activation andco-stimulation markers such as CD80, CD83, and CD86, expression of CCR7allowing migration of DC to draining lymph nodes and facilitatingantigen presentation to T cells, as well as increased secretion ofcytokines such as type I interferons, IL-12, and IL-6. All of thesedownstream events are critical for the induction of an adaptive immuneresponse.

Among the most promising cancer vaccine or immunogenic compositionadjuvants currently in clinical development are the TLR9 agonist CpG andthe synthetic double-stranded RNA (dsRNA) TLR3 ligand poly-ICLC. Inpreclinical studies poly-ICLC appears to be the most potent TLR adjuvantwhen compared to LPS and CpG due to its induction of pro-inflammatorycytokines and lack of stimulation of 1-10, as well as maintenance ofhigh levels of co-stimulatory molecules in DCs1. Furthermore, poly-ICLCwas recently directly compared to CpG in non-human primates (rhesusmacaques) as adjuvant for a protein vaccine or immunogenic compositionconsisting of human papillomavirus (HPV)16 capsomers (Stahl-Hennig C,Eisenblatter M, Jasny E, et al. Synthetic double-stranded RNAs areadjuvants for the induction of T helper 1 and humoral immune responsesto human papillomavirus in rhesus macaques. PLoS pathogens. April 2009;5(4)).

CpG immuno stimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine or immunogenic compositionsetting. Without being bound by theory, CpG oligonucleotides act byactivating the innate (non-adaptive) immune system via Toll-likereceptors (TLR), mainly TLR9. CpG triggered TLR9 activation enhancesantigen-specific humoral and cellular responses to a wide variety ofantigens, including peptide or protein antigens, live or killed viruses,dendritic cell vaccines, autologous cellular vaccines and polysaccharideconjugates in both prophylactic and therapeutic vaccines. Moreimportantly, it enhances dendritic cell maturation and differentiation,resulting in enhanced activation of Th1 cells and strong cytotoxicT-lymphocyte (CTL) generation, even in the absence of CD4 T-cell help.The Th1 bias induced by TLR9 stimulation is maintained even in thepresence of vaccine adjuvants such as alum or incomplete Freund'sadjuvant (IFA) that normally promote a Th2 bias. CpG oligonucleotidesshow even greater adjuvant activity when formulated or co-administeredwith other adjuvants or in formulations such as microparticles, nanoparticles, lipid emulsions or similar formulations, which are especiallynecessary for inducing a strong response when the antigen is relativelyweak. They also accelerate the immune response and enabled the antigendoses to be reduced by approximately two orders of magnitude, withcomparable antibody responses to the full-dose vaccine without CpG insome experiments (Arthur M. Krieg, Nature Reviews, Drug Discovery, 5,Jun. 2006, 471-484). U.S. Pat. No. 6,406,705 B1 describes the combineduse of CpG oligonucleotides, non-nucleic acid adjuvants and an antigento induce an antigen-specific immune response. A commercially availableCpG TLR9 antagonist is dSLIM (double Stem Loop Immunomodulator) byMologen (Berlin, GERMANY), which is a preferred component of thepharmaceutical composition of the present invention. Other TLR bindingmolecules such as RNA binding TLR 7, TLR 8 and/or TLR 9 may also beused.

Other examples of useful adjuvants include, but are not limited to,chemically modified CpGs (e.g. CpR, Idera), Poly(I:CXe.g. polyi:CI2U),non-CpG bacterial DNA or RNA as well as immunoactive small molecules andantibodies such as cyclophosphamide, sunitinib, bevacizumab, celebrex,NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999,CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, andSC58175, which may act therapeutically and/or as an adjuvant. Theamounts and concentrations of adjuvants and additives useful in thecontext of the present invention can readily be determined by theskilled artisan without undue experimentation. Additional adjuvantsinclude colony-stimulating factors, such as Granulocyte MacrophageColony Stimulating Factor (GM-CSF, sargramostim).

Poly-ICLC is a synthetically prepared double-stranded RNA consisting ofpolyI and polyC strands of average length of about 5000 nucleotides,which has been stabilized to thermal denaturation and hydrolysis byserum nucleases by the addition of polylysine andcarboxymethylcellulose. The compound activates TLR3 and the RNAhelicase-domain of MDA5, both members of the PAMP family, leading to DCand natural killer (NK) cell activation and production of a “naturalmix” of type I interferons, cytokines, and chemokines. Furthermore,poly-ICLC exerts a more direct, broad host-targeted anti-infectious andpossibly antitumor effect mediated by the two IFN-inducible nuclearenzyme systems, the 2′5′-OAS and the P1/eIF2a kinase, also known as thePKR (4-6), as well as RIG-I helicase and MDA5.

In rodents and non-human primates, poly-ICLC was shown to enhance T cellresponses to viral antigens, cross-priming, and the induction of tumor-,virus-, and autoantigen-specific CD8+ T-cells. In a recent study innon-human primates, poly-ICLC was found to be essential for thegeneration of antibody responses and T-cell immunity to DC targeted ornon-targeted HIV Gag p24 protein, emphasizing its effectiveness as avaccine adjuvant.

In human subjects, transcriptional analysis of serial whole bloodsamples revealed similar gene expression profiles among the 8 healthyhuman volunteers receiving one single s.c. administration of poly-ICLCand differential expression of up to 212 genes between these 8 subjectsversus 4 subjects receiving placebo. Remarkably, comparison of thepoly-ICLC gene expression data to previous data from volunteersimmunized with the highly effective yellow fever vaccine YF17D showedthat a large number of transcriptional and signal transduction canonicalpathways, including those of the innate immune system, were similarlyupregulated at peak time points.

More recently, an immunologic analysis was reported on patients withovarian, fallopian tube, and primary peritoneal cancer in second orthird complete clinical remission who were treated on a phase 1 study ofsubcutaneous vaccination with synthetic overlapping long peptides (OLP)from the cancer testis antigen NY-ESO-1 alone or with Montanide-ISA-51,or with 1.4 mg poly-ICLC and Montanide. The generation ofNY-ESO-1-specific CD4+ and CD8+ T-cell and antibody responses weremarkedly enhanced with the addition of poly-ICLC and Montanide comparedto OLP alone or OLP and Montanide.

A vaccine or immunogenic composition according to the present inventionmay comprise more than one different adjuvant. Furthermore, theinvention encompasses a therapeutic composition comprising any adjuvantsubstance including any of those herein discussed. It is alsocontemplated that the peptide or polypeptide, and the adjuvant can beadministered separately in any appropriate sequence.

A carrier may be present independently of an adjuvant. The carrier maybe covalently linked to the antigen. A carrier can also be added to theantigen by inserting DNA encoding the carrier in frame with DNA encodingthe antigen. The function of a carrier can for example be to conferstability, to increase the biological activity, or to increase serumhalf-life. Extension of the half-life can help to reduce the number ofapplications and to lower doses, thus are beneficial for therapeutic butalso economic reasons. Furthermore, a carrier may aid presentingpeptides to T-cells. The carrier may be any suitable carrier known tothe person skilled in the art, for example a protein or an antigenpresenting cell. A carrier protein could be but is not limited tokeyhole limpet hemocyanin, serum proteins such as transferrin, bovineserum albumin, human serum albumin, thyroglobulin or ovalbumin,immunoglobulins, or hormones, such as insulin or palmitic acid. Forimmunization of humans, the carrier may be a physiologically acceptablecarrier acceptable to humans and safe. However, tetanus toxoid and/ordiptheria toxoid are suitable carriers in one embodiment of theinvention. Alternatively, the carrier may be dextrans for examplesepharose.

Cytotoxic T-cells (CTLs) recognize an antigen in the form of a peptidebound to an MHC molecule rather than the intact foreign antigen itself.The MHC molecule itself is located at the cell surface of an antigenpresenting cell. Thus, an activation of CTLs is only possible if atrimeric complex of peptide antigen, MHC molecule, and APC is present.Correspondingly, it may enhance the immune response if not only thepeptide is used for activation of CTLs, but if additionally APCs withthe respective MHC molecule are added. Therefore, in some embodimentsthe vaccine or immunogenic composition according to the presentinvention additionally contains at least one antigen presenting cell.

The antigen-presenting cell (or stimulator cell) typically has an MHCclass I or II molecule on its surface, and in one embodiment issubstantially incapable of itself loading the MHC class I or II moleculewith the selected antigen. As is described in more detail herein, theMHC class I or II molecule may readily be loaded with the selectedantigen in vitro.

CD8+ cell activity may be augmented through the use of CD4+ cells. Theidentification of CD4 T+ cell epitopes for tumor antigens has attractedinterest because many immune based therapies against cancer may be moreeffective if both CD8+ and CD4+ T lymphocytes are used to target apatient's tumor. CD4+ cells are capable of enhancing CD8 T cellresponses. Many studies in animal models have clearly demonstratedbetter results when both CD4+ and CD8+ T cells participate in anti-tumorresponses (see e.g., Nishimura et al. (1999) Distinct role ofantigen-specific T helper type 1 (TH1) and Th2 cells in tumoreradication in vivo. J Ex Med 190:617-27). Universal CD4+ T cellepitopes have been identified that are applicable to developingtherapies against different types of cancer (see e.g., Kobayashi et al.(2008) Current Opinion in Immunology 20:221-27). For example, an HLA-DRrestricted helper peptide from tetanus toxoid was used in melanomavaccines to activate CD4+ T cells non-specifically (see e.g., Slingluffet al. (2007) Immunologic and Clinical Outcomes of a Randomized Phase IITrial of Two Multipeptide Vaccines for Melanoma in the Adjuvant Setting,Clinical Cancer Research 13(21):6386-95). It is contemplated within thescope of the invention that such CD4+ cells may be applicable at threelevels that vary in their tumor specificity: 1) a broad level in whichuniversal CD4+ epitopes (e.g., tetanus toxoid) may be used to augmentCD8+ cells; 2) an intermediate level in which native, tumor-associatedCD4+ epitopes may be used to augment CD8+ cells; and 3) a patientspecific level in which neoantigen CD4+ epitopes may be used to augmentCD8+ cells in a patient specific manner.

CD8+ cell immunity may also be generated with neoantigen loadeddendritic cell (DC) vaccine. DCs are potent antigen-presenting cellsthat initiate T cell immunity and can be used as cancer vaccines whenloaded with one or more peptides of interest, for example, by directpeptide injection. For example, patients that were newly diagnosed withmetastatic melanoma were shown to be immunized against 3HLA-A*0201-restricted gp100 melanoma antigen-derived peptides withautologous peptide pulsed CD40L/IFN-g-activated mature DCs via anIL-12p70-producing patient DC vaccine (see e.g., Carreno et al (2013)L-12p70-producing patient DC vaccine elicits Tc1-polarized immunity,Journal of Clinical Investigation, 123(8):3383-94 and Ali et al. (2009)In situ regulation of DC subsets and T cells mediates tumor regressionin mice, Cancer Immunotherapy, 1(8): 1-10). It is contemplated withinthe scope of the invention that neoantigen loaded DCs may be preparedusing the synthetic TLR 3 agonist Polyinosinic-PolycytidylicAcid-poly-L-lysine Carboxymethylcellulose (Poly-ICLC) to stimulate theDCs. Poly-ICLC is a potent individual maturation stimulus for human DCsas assessed by an upregulation of CD83 and CD86, induction ofinterleukin-12 (IL-12), tumor necrosis factor (TNF), interferongamma-induced protein 10 (IP-10), interleukin 1 (IL-1), and type Iinterferons (IFN), and minimal interleukin 10 (IL-10) production. DCsmay be differentiated from frozen peripheral blood mononuclear cells(PBMCs) obtained by leukapheresis, while PBMCs may be isolated by Ficollgradient centrifugation and frozen in aliquots.

Illustratively, the following 7 day activation protocol may be used. Day1-PBMCs are thawed and plated onto tissue culture flasks to select formonocytes which adhere to the plastic surface after 1-2 hr incubation at37° C. in the tissue culture incubator. After incubation, thelymphocytes are washed off and the adherent monocytes are cultured for 5days in the presence of interleukin-4 (IL-4) and granulocytemacrophage-colony stimulating factor (GM-CSF) to differentiate toimmature DCs. On Day 6, immature DCs are pulsed with the keyhole limpethemocyanin (KLH) protein which serves as a control for the quality ofthe vaccine and may boost the immunogenicity of the vaccine. The DCs arestimulated to mature, loaded with peptide antigens, and incubatedovernight. On Day 7, the cells are washed, and frozen in 1 ml aliquotscontaining 4-20×10⁶ cells using a controlled-rate freezer. Lot releasetesting for the batches of DCs may be performed to meet minimumspecifications before the DCs are injected into patients (see e.g.,Sabado et al. (2013) Preparation of tumor antigen-loaded maturedendritic cells for immunotherapy, J. Vis Exp. August 1; (78). doi:10.3791/50085).

A DC vaccine may be incorporated into a scaffold system to facilitatedelivery to a patient. Therapeutic treatment of a patients neoplasiawith a DC vaccine may utilize a biomaterial system that releases factorsthat recruit host dendritic cells into the device, differentiates theresident, immature DCs by locally presenting adjuvants (e.g., dangersignals) while releasing antigen, and promotes the release of activated,antigen loaded DCs to the lymph nodes (or desired site of action) wherethe DCs may interact with T cells to generate a potent cytotoxic Tlymphocyte response to the cancer neoantigens. Implantable biomaterialsmay be used to generate a potent cytotoxic T lymphocyte response againsta neoplasia in a patient specific manner. The biomaterial-residentdendritic cells may then be activated by exposing them to danger signalsmimicking infection, in concert with release of antigen from thebiomaterial. The activated dendritic cells then migrate from thebiomaterials to lymph nodes to induce a cytotoxic T effector response.This approach has previously been demonstrated to lead to regression ofestablished melanoma in preclinical studies using a lysate prepared fromtumor biopsies (see e.g., Ali et al. (2209) In situ regulation of DCsubsets and T cells mediates tumor regression in mice, CancerImmunotherapy 1(8):1-10; Ali et al. (2009) Infection-mimicking materialsto program dendritic cells in situ. Nat Mater 8:151-8), and such avaccine is currently being tested in a Phase I clinical trial recentlyinitiated at the Dana-Farber Cancer Institute. This approach has alsobeen shown to lead to regression of glioblastoma, as well as theinduction of a potent memory response to prevent relapse, using the C6rat glioma model.24 in the current proposal. The ability of such animplantable, biomatrix vaccine delivery scaffold to amplify and sustaintumor specific dendritic cell activation may lead to more robustanti-tumor immunosensitization than can be achieved by traditionalsubcutaneous or intra-nodal vaccine administrations.

Preferably, the antigen presenting cells are dendritic cells. Suitably,the dendritic cells are autologous dendritic cells that are pulsed withthe neoantigenic peptide. The peptide may be any suitable peptide thatgives rise to an appropriate T-cell response. T-cell therapy usingautologous dendritic cells pulsed with peptides from a tumor associatedantigen is disclosed in Murphy et al. (1996) The Prostate 29, 371-380and Tjua et al. (1997) The Prostate 32, 272-278.

Thus, in one embodiment of the present invention the vaccine orimmunogenic composition containing at least one antigen presenting cellis pulsed or loaded with one or more peptides of the present invention.Alternatively, peripheral blood mononuclear cells (PBMCs) isolated froma patient may be loaded with peptides ex vivo and injected back into thepatient. As an alternative the antigen presenting cell comprises anexpression construct encoding a peptide of the present invention. Thepolynucleotide may be any suitable polynucleotide and it is preferredthat it is capable of transducing the dendritic cell, thus resulting inthe presentation of a peptide and induction of immunity.

The inventive pharmaceutical composition may be compiled so that theselection, number and/or amount of peptides present in the compositionis/are tissue, cancer, and/or patient-specific. For instance, the exactselection of peptides can be guided by expression patterns of the parentproteins in a given tissue to avoid side effects. The selection may bedependent on the specific type of cancer, the status of the disease,earlier treatment regimens, the immune status of the patient, and, ofcourse, the HLA-haplotype of the patient. Furthermore, the vaccine orimmunogenic composition according to the invention can containindividualized components, according to personal needs of the particularpatient. Examples include varying the amounts of peptides according tothe expression of the related neoantigen in the particular patient,unwanted side-effects due to personal allergies or other treatments, andadjustments for secondary treatments following a first round or schemeof treatment.

Pharmaceutical compositions comprising the peptide of the invention maybe administered to an individual already suffering from cancer. Intherapeutic applications, compositions are administered to a patient inan amount sufficient to elicit an effective CTL response to the tumorantigen and to cure or at least partially arrest symptoms and/orcomplications. An amount adequate to accomplish this is defined as“therapeutically effective dose.” Amounts effective for this use candepend on, e.g., the peptide composition, the manner of administration,the stage and severity of the disease being treated, the weight andgeneral state of health of the patient, and the judgment of theprescribing physician, but generally range for the initial immunization(that is for therapeutic or prophylactic administration) from about 1.0μg to about 50,000 μg of peptide for a 70 kg patient, followed byboosting dosages or from about 1.0 μg to about 10,000 μg of peptidepursuant to a boosting regimen over weeks to months depending upon thepatient's response and condition and possibly by measuring specific CTLactivity in the patient's blood. It should be kept in mind that thepeptide and compositions of the present invention may generally beemployed in serious disease states, that is, life-threatening orpotentially life threatening situations, especially when the cancer hasmetastasized. For therapeutic use, administration should begin as soonas possible after the detection or surgical removal of tumors. This isfollowed by boosting doses until at least symptoms are substantiallyabated and for a period thereafter.

The pharmaceutical compositions (e.g., vaccine compositions) fortherapeutic treatment are intended for parenteral, topical, nasal, oralor local administration. Preferably, the pharmaceutical compositions areadministered parenterally, e.g., intravenously, subcutaneously,intradermally, or intramuscularly. The compositions may be administeredat the site of surgical excision to induce a local immune response tothe tumor. The invention provides compositions for parenteraladministration which comprise a solution of the peptides and vaccine orimmunogenic compositions are dissolved or suspended in an acceptablecarrier, preferably an aqueous carrier. A variety of aqueous carriersmay be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine,hyaluronic acid and the like. These compositions may be sterilized byconventional, well known sterilization techniques, or may be sterilefiltered. The resulting aqueous solutions may be packaged for use as is,or lyophilized, the lyophilized preparation being combined with asterile solution prior to administration. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc.

A liposome suspension containing a peptide may be administeredintravenously, locally, topically, etc. in a dose which varies accordingto, inter alia, the manner of administration, the peptide beingdelivered, and the stage of the disease being treated. For targeting tothe immune cells, a ligand, such as, e.g., antibodies or fragmentsthereof specific for cell surface determinants of the desired immunesystem cells, can be incorporated into the liposome.

For solid compositions, conventional or nanoparticle nontoxic solidcarriers may be used which include, for example, pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharin,talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.For oral administration, a pharmaceutically acceptable nontoxiccomposition is formed by incorporating any of the normally employedexcipients, such as those carriers previously listed, and generally10-95% of active ingredient, that is, one or more peptides of theinvention, and more preferably at a concentration of 25%-75%.

For aerosol administration, the immunogenic peptides are preferablysupplied in finely divided form along with a surfactant and propellant.Typical percentages of peptides are 0.01%-20% by weight, preferably1%-10%. The surfactant can, of course, be nontoxic, and preferablysoluble in the propellant. Representative of such agents are the estersor partial esters of fatty acids containing from 6 to 22 carbon atoms,such as caproic, octanoic, lauric, palmitic, stearic, linoleic,linolenic, olesteric and oleic acids with an aliphatic polyhydricalcohol or its cyclic anhydride. Mixed esters, such as mixed or naturalglycerides may be employed. The surfactant may constitute 0.1%-20% byweight of the composition, preferably 0.25-5%. The balance of thecomposition is ordinarily propellant. A carrier can also be included asdesired, as with, e.g., lecithin for intranasal delivery.

The peptides and polypeptides of the invention can be readilysynthesized chemically utilizing reagents that are free of contaminatingbacterial or animal substances (Merrifield R B: Solid phase peptidesynthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc.85:2149-54, 1963).

The peptides and polypeptides of the invention can also be expressed bya vector, e.g., a nucleic acid molecule as herein-discussed, e.g., RNAor a DNA plasmid, a viral vector such as a poxvirus, e.g., orthopoxvirus, avipox virus, or adenovirus, AAV or lentivirus. This approachinvolves the use of a vector to express nucleotide sequences that encodethe peptide of the invention. Upon introduction into an acutely orchronically infected host or into a noninfected host, the vectorexpresses the immunogenic peptide, and thereby elicits a host CTLresponse.

For therapeutic or immunization purposes, nucleic acids encoding thepeptide of the invention and optionally one or more of the peptidesdescribed herein can also be administered to the patient. A number ofmethods are conveniently used to deliver the nucleic acids to thepatient. For instance, the nucleic acid can be delivered directly, as“naked DNA”. This approach is described, for instance, in Wolff et al.,Science 247: 1465-1468 (1990) as well as U.S. Pat. Nos. 5,580,859 and5,589,466. The nucleic acids can also be administered using ballisticdelivery as described, for instance, in U.S. Pat. No. 5,204,253.Particles comprised solely of DNA can be administered. Alternatively,DNA can be adhered to particles, such as gold particles. Generally, aplasmid for a vaccine or immunological composition can comprise DNAencoding an antigen (e.g., one or more neoantigens) operatively linkedto regulatory sequences which control expression or expression andsecretion of the antigen from a host cell, e.g., a mammalian cell; forinstance, from upstream to downstream, DNA for a promoter, such as amammalian virus promoter (e.g., a CMV promoter such as an hCMV or mCMVpromoter, e.g., an early-intermediate promoter, or an SV40 promoter—seedocuments cited or incorporated herein for useful promoters), DNA for aeukaryotic leader peptide for secretion (e.g., tissue plasminogenactivator), DNA for the neoantigen(s), and DNA encoding a terminator(e.g., the 3′ UTR transcriptional terminator from the gene encodingBovine Growth Hormone or bGH polyA). A composition can contain more thanone plasmid or vector, whereby each vector contains and expresses adifferent neoantigen. Mention is also made of Wasmoen U.S. Pat. No.5,849,303, and Dale U.S. Pat. No. 5,811,104, whose text may be useful.DNA or DNA plasmid formulations can be formulated with or insidecationic lipids; and, as to cationic lipids, as well as adjuvants,mention is also made of Loosmore U.S. Patent Application 2003/0104008.Also, teachings in Audonnet U.S. Pat. Nos. 6,228,846 and 6,159,477 maybe relied upon for DNA plasmid teachings that can be employed inconstructing and using DNA plasmids that contain and express in vivo.

The nucleic acids can also be delivered complexed to cationic compounds,such as cationic lipids. Lipid-mediated gene delivery methods aredescribed, for instance, in WO1996/18372; WO 1993/24640; Mannino &Gould-Fogerite, BioTechniques 6(7): 682-691 (1988); U.S. Pat. No.5,279,833; WO 1991/06309; and Feigner et al., Proc. Natl. Acad. Sci. USA84: 7413-7414 (1987).

RNA encoding the peptide of interest (e.g., mRNA) can also be used fordelivery (see, e.g., Kiken et al, 2011; Su et al, 2011; see also U.S.Pat. No. 8,278,036; Halabi et al. J Clin Oncol (2003) 21:1232-1237;Petsch et al, Nature Biotechnology 2012 Dec. 7; 30(12): 1210-6).

Information concerning poxviruses that may be used in the practice ofthe invention, such as Chordopoxvirinae subfamily poxviruses (poxvirusesof vertebrates), for instance, orthopoxviruses and avipoxviruses, e.g.,vaccinia virus (e.g., Wyeth Strain, WR Strain (e.g., ATCC® VR-1354),Copenhagen Strain, NYVAC, NYVAC.1, NYVAC.2, MVA, MVA-BN), canarypoxvirus (e.g., Wheatley C93 Strain, ALVAC), fowlpox virus (e.g., FP9Strain, Webster Strain, TROVAC), dovepox, pigeonpox, quailpox, andraccoon pox, inter alia, synthetic or non-naturally occurringrecombinants thereof, uses thereof, and methods for making and usingsuch recombinants may be found in scientific and patent literature, suchas:

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As to adenovirus vectors useful in the practice of the invention,mention is made of U.S. Pat. No. 6,955,808. The adenovirus vector usedcan be selected from the group consisting of the Ad5, Ad35, Ad11, C6,and C7 vectors. The sequence of the Adenovirus 5 (“Ad5”) genome has beenpublished. (Chroboczek, J., Bieber, F., and Jacrot, B. (1992) TheSequence of the Genome of Adenovirus Type 5 and Its Comparison with theGenome of Adenovirus Type 2, Virology 186, 280-285; the contents ifwhich is hereby incorporated by reference). Ad35 vectors are describedin U.S. Pat. Nos. 6,974,695, 6,913,922, and 6,869,794. Ad11 vectors aredescribed in U.S. Pat. No. 6,913,922. C6 adenovirus vectors aredescribed in U.S. Pat. Nos. 6,780,407; 6,537,594; 6,309,647; 6,265,189;6,156,567; 6,090,393; 5,942,235 and 5,833,975. C7 vectors are describedin U.S. Pat. No. 6,277,558. Adenovirus vectors that are E1-defective ordeleted, E3-defective or deleted, and/or E4-defective or deleted mayalso be used. Certain adenoviruses having mutations in the E1 regionhave improved safety margin because E1-defective adenovirus mutants arereplication-defective in non-permissive cells, or, at the very least,are highly attenuated. Adenoviruses having mutations in the E3 regionmay have enhanced the immunogenicity by disrupting the mechanism wherebyadenovirus down-regulates MHC class I molecules. Adenoviruses having E4mutations may have reduced immunogenicity of the adenovirus vectorbecause of suppression of late gene expression. Such vectors may beparticularly useful when repeated re-vaccination utilizing the samevector is desired. Adenovirus vectors that are deleted or mutated in E1,E3, E4, E1 and E3, and E1 and E4 can be used in accordance with thepresent invention. Furthermore, “gutless” adenovirus vectors, in whichall viral genes are deleted, can also be used in accordance with thepresent invention. Such vectors require a helper virus for theirreplication and require a special human 293 cell line expressing bothE1a and Cre, a condition that does not exist in natural environment.Such “gutless” vectors are non-immunogenic and thus the vectors may beinoculated multiple times for re-vaccination. The “gutless” adenovirusvectors can be used for insertion of heterologous inserts/genes such asthe transgenes of the present invention, and can even be used forco-delivery of a large number of heterologous inserts/genes.

As to lentivirus vector systems useful in the practice of the invention,mention is made of U.S. Pat. Nos. 6,428,953, 6,165,782, 6,013,516,5,994,136, 6,312,682, and 7,198,784, and documents cited therein.

With regard to AAV vectors useful in the practice of the invention,mention is made of U.S. Pat. Nos. 5,658,785, 7,115,391, 7,172,893,6,953,690, 6,936,466, 6,924,128, 6,893,865, 6,793,926, 6,537,540,6,475,769 and 6,258,595, and documents cited therein.

Another vector is BCG (Bacille Calmette Guerin). BCG vectors aredescribed in Stover et al. (Nature 351:456-460 (1991)). A wide varietyof other vectors useful for therapeutic administration or immunizationof the peptides of the invention, e.g., Salmonella typhi vectors and thelike, is apparent to those skilled in the art from the descriptionherein.

Vectors can be administered so as to have in vivo expression andresponse akin to doses and/or responses elicited by antigenadministration

A preferred means of administering nucleic acids encoding the peptide ofthe invention uses minigene constructs encoding multiple epitopes. Tocreate a DNA sequence encoding the selected CTL epitopes (minigene) forexpression in human cells, the amino acid sequences of the epitopes arereverse translated. A human codon usage table is used to guide the codonchoice for each amino acid. These epitope-encoding DNA sequences aredirectly adjoined, creating a continuous polypeptide sequence. Tooptimize expression and/or immunogenicity, additional elements can beincorporated into the minigene design. Examples of amino acid sequencethat could be reverse translated and included in the minigene sequenceinclude: helper T lymphocyte, epitopes, a leader (signal) sequence, andan endoplasmic reticulum retention signal. In addition, MHC presentationof CTL epitopes may be improved by including synthetic (e.g.poly-alanine) or naturally-occurring flanking sequences adjacent to theCTL epitopes.

The minigene sequence is converted to DNA by assembling oligonucleotidesthat encode the plus and minus strands of the minigene. Overlappingoligonucleotides (30-100 bases long) are synthesized, phosphorylated,purified and annealed under appropriate conditions using well knowntechniques. The ends of the oligonucleotides are joined using T4 DNAligase. This synthetic minigene, encoding the CTL epitope polypeptide,can then cloned into a desired expression vector.

Standard regulatory sequences well known to those of skill in the artare included in the vector to ensure expression in the target cells.Several vector elements are required: a promoter with a down-streamcloning site for minigene insertion; a polyadenylation signal forefficient transcription termination; an E. coli origin of replication;and an E. coli selectable marker (e.g. ampicillin or kanamycinresistance). Numerous promoters can be used for this purpose, e.g., thehuman cytomegalovirus (hCMV) promoter. See, U.S. Pat. Nos. 5,580,859 and5,589,466 for other suitable promoter sequences.

Additional vector modifications may be desired to optimize minigeneexpression and immunogenicity. In some cases, introns are required forefficient gene expression, and one or more synthetic ornaturally-occurring introns could be incorporated into the transcribedregion of the minigene. The inclusion of mRNA stabilization sequencescan also be considered for increasing minigene expression. It hasrecently been proposed that immuno stimulatory sequences (ISSs or CpGs)play a role in the immunogenicity of DNA' vaccines. These sequencescould be included in the vector, outside the minigene coding sequence,if found to enhance immunogenicity.

In some embodiments, a bicistronic expression vector, to allowproduction of the minigene-encoded epitopes and a second proteinincluded to enhance or decrease immunogenicity can be used. Examples ofproteins or polypeptides that could beneficially enhance the immuneresponse if co-expressed include cytokines (e.g., IL2, IL12, GM-CSF),cytokine-inducing molecules (e.g. LeIF) or costimulatory molecules.Helper (HTL) epitopes could be joined to intracellular targeting signalsand expressed separately from the CTL epitopes. This would allowdirection of the HTL epitopes to a cell compartment different than theCTL epitopes. If required, this could facilitate more efficient entry ofHTL epitopes into the MHC class II pathway, thereby improving CTLinduction. In contrast to CTL induction, specifically decreasing theimmune response by co-expression of immunosuppressive molecules (e.g.TGF-β) may be beneficial in certain diseases.

Once an expression vector is selected, the minigene is cloned into thepolylinker region downstream of the promoter. This plasmid istransformed into an appropriate E. coli strain, and DNA is preparedusing standard techniques. The orientation and DNA sequence of theminigene, as well as all other elements included in the vector, areconfirmed using restriction mapping and DNA sequence analysis. Bacterialcells harboring the correct plasmid can be stored as a master cell bankand a working cell bank.

Purified plasmid DNA can be prepared for injection using a variety offormulations. The simplest of these is reconstitution of lyophilized DNAin sterile phosphate-buffer saline (PBS). A variety of methods have beendescribed, and new techniques may become available. As noted herein,nucleic acids are conveniently formulated with cationic lipids. Inaddition, glycolipids, fusogenic liposomes, peptides and compoundsreferred to collectively as protective, interactive, non-condensing(PINC) could also be complexed to purified plasmid DNA to influencevariables such as stability, intramuscular dispersion, or trafficking tospecific organs or cell types.

Target cell sensitization can be used as a functional assay forexpression and MHC class I presentation of minigene-encoded CTLepitopes. The plasmid DNA is introduced into a mammalian cell line thatis suitable as a target for standard CTL chromium release assays. Thetransfection method used is dependent on the final formulation.Electroporation can be used for “naked” DNA, whereas cationic lipidsallow direct in vitro transfection. A plasmid expressing greenfluorescent protein (GFP) can be co-transfected to allow enrichment oftransfected cells using fluorescence activated cell sorting (FACS).These cells are then chromium-51 labeled and used as target cells forepitope-specific CTL lines. Cytolysis, detected by 51 Cr release,indicates production of MHC presentation of mini gene-encoded CTLepitopes.

In vivo immunogenicity is a second approach for functional testing ofminigene DNA formulations. Transgenic mice expressing appropriate humanMHC molecules are immunized with the DNA product. The dose and route ofadministration are formulation dependent (e.g. IM for DNA in PBS, IP forlipid-complexed DNA). Twenty-one days after immunization, splenocytesare harvested and restimulated for 1 week in the presence of peptidesencoding each epitope being tested. These effector cells (CTLs) areassayed for cytolysis of peptide-loaded, chromium-51 labeled targetcells using standard techniques. Lysis of target cells sensitized by MHCloading of peptides corresponding to minigene-encoded epitopesdemonstrates DNA vaccine function for in vivo induction of CTLs.

Peptides may be used to elicit CTL ex vivo, as well. The resulting CTL,can be used to treat chronic tumors in patients in need thereof that donot respond to other conventional forms of therapy, or does not respondto a peptide vaccine approach of therapy. Ex vivo CTL responses to aparticular tumor antigen are induced by incubating in tissue culture thepatient's CTL precursor cells (CTLp) together with a source ofantigen-presenting cells (APC) and the appropriate peptide. After anappropriate incubation time (typically 1-4 weeks), in which the CTLp areactivated and mature and expand into effector CTL, the cells are infusedback into the patient, where they destroy their specific target cell(i.e., a tumor cell). In order to optimize the in vitro conditions forthe generation of specific cytotoxic T cells, the culture of stimulatorcells are maintained in an appropriate serum-free medium.

Prior to incubation of the stimulator cells with the cells to beactivated, e.g., precursor CD8+ cells, an amount of antigenic peptide isadded to the stimulator cell culture, of sufficient quantity to becomeloaded onto the human Class I molecules to be expressed on the surfaceof the stimulator cells. In the present invention, a sufficient amountof peptide is an amount that allows about 200, and preferably 200 ormore, human Class I MHC molecules loaded with peptide to be expressed onthe surface of each stimulator cell. Preferably, the stimulator cellsare incubated with >2 μg/ml peptide. For example, the stimulator cellsare incubates with >3, 4, 5, 10, 15, or more μg/ml peptide.

Resting or precursor CD8+ cells are then incubated in culture with theappropriate stimulator cells for a time period sufficient to activatethe CD8+ cells. Preferably, the CD8+ cells are activated in anantigen-specific manner. The ratio of resting or precursor CD8+(effector) cells to stimulator cells may vary from individual toindividual and may further depend upon variables such as the amenabilityof an individual's lymphocytes to culturing conditions and the natureand severity of the disease condition or other condition for which thewithin-described treatment modality is used. Preferably, however, thelymphocyte:stimulator cell ratio is in the range of about 30:1 to 300:1.The effector/stimulator culture may be maintained for as long a time asis necessary to stimulate a therapeutically useable or effective numberof CD8+ cells.

The induction of CTL in vitro requires the specific recognition ofpeptides that are bound to allele specific MHC class I molecules on APC.The number of specific MHC/peptide complexes per APC is crucial for thestimulation of CTL, particularly in primary immune responses. Whilesmall amounts of peptide/MHC complexes per cell are sufficient to rendera cell susceptible to lysis by CTL, or to stimulate a secondary CTLresponse, the successful activation of a CTL precursor (pCTL) duringprimary response requires a significantly higher number of MHC/peptidecomplexes. Peptide loading of empty major histocompatability complexmolecules on cells allows the induction of primary cytotoxic Tlymphocyte responses.

Since mutant cell lines do not exist for every human MHC allele, it isadvantageous to use a technique to remove endogenous MHC-associatedpeptides from the surface of APC, followed by loading the resultingempty MHC molecules with the immunogenic peptides of interest. The useof non-transformed (non-tumorigenic), noninfected cells, and preferably,autologous cells of patients as APC is desirable for the design of CTLinduction protocols directed towards development of ex vivo CTLtherapies. This application discloses methods for stripping theendogenous MHC-associated peptides from the surface of APC followed bythe loading of desired peptides.

A stable MHC class I molecule is a trimeric complex formed of thefollowing elements: 1) a peptide usually of 8-10 residues, 2) atransmembrane heavy polymorphic protein chain which bears thepeptide-binding site in its a1 and a2 domains, and 3) a non-covalentlyassociated non-polymorphic light chain, p2microglobulin. Removing thebound peptides and/or dissociating the p2microglobulin from the complexrenders the MHC class I molecules nonfunctional and unstable, resultingin rapid degradation. All MHC class I molecules isolated from PBMCs haveendogenous peptides bound to them. Therefore, the first step is toremove all endogenous peptides bound to MHC class I molecules on the APCwithout causing their degradation before exogenous peptides can be addedto them.

Two possible ways to free up MHC class I molecules of bound peptidesinclude lowering the culture temperature from 37° C. to 26° C. overnightto destablize p2microglobulin and stripping the endogenous peptides fromthe cell using a mild acid treatment. The methods release previouslybound peptides into the extracellular environment allowing new exogenouspeptides to bind to the empty class I molecules. The cold-temperatureincubation method enables exogenous peptides to bind efficiently to theMHC complex, but requires an overnight incubation at 26° C. which mayslow the cell's metabolic rate. It is also likely that cells notactively synthesizing MHC molecules (e.g., resting PBMC) would notproduce high amounts of empty surface MHC molecules by the coldtemperature procedure.

Harsh acid stripping involves extraction of the peptides withtrifluoroacetic acid, pH 2, or acid denaturation of the immunoaffinitypurified class I-peptide complexes. These methods are not feasible forCTL induction, since it is important to remove the endogenous peptideswhile preserving APC viability and an optimal metabolic state which iscritical for antigen presentation. Mild acid solutions of pH 3 such asglycine or citrate-phosphate buffers have been used to identifyendogenous peptides and to identify tumor associated T cell epitopes.The treatment is especially effective, in that only the MHC class Imolecules are destabilized (and associated peptides released), whileother surface antigens remain intact, including MHC class II molecules.Most importantly, treatment of cells with the mild acid solutions do notaffect the cell's viability or metabolic state. The mild acid treatmentis rapid since the stripping of the endogenous peptides occurs in twominutes at 4° C. and the APC is ready to perform its function after theappropriate peptides are loaded. The technique is utilized herein tomake peptide-specific APCs for the generation of primaryantigen-specific CTL. The resulting APC are efficient in inducingpeptide-specific CD8+ CTL.

Activated CD8+ cells may be effectively separated from the stimulatorcells using one of a variety of known methods. For example, monoclonalantibodies specific for the stimulator cells, for the peptides loadedonto the stimulator cells, or for the CD8+ cells (or a segment thereof)may be utilized to bind their appropriate complementary ligand.Antibody-tagged molecules may then be extracted from thestimulator-effector cell admixture via appropriate means, e.g., viawell-known immunoprecipitation or immunoassay methods.

Effective, cytotoxic amounts of the activated CD8+ cells can varybetween in vitro and in vivo uses, as well as with the amount and typeof cells that are the ultimate target of these killer cells. The amountcan also vary depending on the condition of the patient and should bedetermined via consideration of all appropriate factors by thepractitioner. Preferably, however, about 1×10⁶ to about 1×10¹², morepreferably about 1×10⁸ to about 1×10¹¹, and even more preferably, about1×10⁹ to about 1×10¹⁰ activated CD8+ cells are utilized for adulthumans, compared to about 5×10⁶-5×10⁷ cells used in mice.

Preferably, as discussed herein, the activated CD8+ cells are harvestedfrom the cell culture prior to administration of the CD8+ cells to theindividual being treated. It is important to note, however, that unlikeother present and proposed treatment modalities, the present method usesa cell culture system that is not tumorigenic. Therefore, if completeseparation of stimulator cells and activated CD8+ cells are notachieved, there is no inherent danger known to be associated with theadministration of a small number of stimulator cells, whereasadministration of mammalian tumor-promoting cells may be extremelyhazardous.

Methods of re-introducing cellular components are known in the art andinclude procedures such as those exemplified in U.S. Pat. No. 4,844,893to Honsik, et al. and U.S. Pat. No. 4,690,915 to Rosenberg. For example,administration of activated CD8+ cells via intravenous infusion isappropriate.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook,1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture”(Freshney, 1987); “Methods in Enzymology” “Handbook of ExperimentalImmunology” (Wei, 1996); “Gene Transfer Vectors for Mammalian Cells”(Miller and Calos, 1987); “Current Protocols in Molecular Biology”(Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques areapplicable to the production of the polynucleotides and polypeptides ofthe invention, and, as such, may be considered in making and practicingthe invention. Particularly useful techniques for particular embodimentsare discussed in the sections that follow.

Therapeutic Methods

The present invention provides methods of inducing a neoplasia/tumorspecific immune response in a subject, vaccinating against aneoplasia/tumor, treating and or alleviating a symptom of cancer in asubject by administering the subject a neoplasia vaccine or aneoantigenic peptide or composition of the invention.

According to the invention, the herein-described neoplasia vaccine orimmunogenic composition may be used for a patient that has beendiagnosed as having cancer, or at risk of developing cancer. In oneembodiment, the patient may have a solid tumor such as breast, ovarian,prostate, lung, kidney, gastric, colon, testicular, head and neck,pancreas, brain, melanoma, and other tumors of tissue organs andhematological tumors, such as lymphomas and leukemias, including acutemyelogenous leukemia, chronic myelogenous leukemia, chronic lymphocyticleukemia, T cell lymphocytic leukemia, and B cell lymphomas.

The peptide or composition of the invention is administered in an amountsufficient to induce a CTL response.

The herein-described compositions and methods may be used on patients inneed thereof with any cancer according to the general flow process shownin FIG. 2. Patients in need thereof may receive a series of primingvaccinations with a mixture of personalized tumor-specific peptides.Additionally, over a 4 week period the priming may be followed by twoboosts during a maintenance phase. All vaccinations are subcutaneouslydelivered. The vaccine or immunogenic composition is evaluated forsafety, tolerability, immune response and clinical effect in patientsand for feasibility of producing vaccine or immunogenic composition andsuccessfully initiating vaccination within an appropriate time frame.The first cohort can consist of 5 patients, and after safety isadequately demonstrated, an additional cohort of 10 patients may beenrolled. Peripheral blood is extensively monitored for peptide-specificT-cell responses and patients are followed for up to two years to assessdisease recurrence.

Vaccine or Immunogenic Composition Kits and Co-Packaging

In an aspect, the invention provides kits containing any one or more ofthe elements discussed herein to allow administration of the immunogeniccomposition or vaccine. Elements may be provided individually or incombinations, and may be provided in any suitable container, such as avial, a bottle, or a tube. In some embodiments, the kit includesinstructions in one or more languages, for example in more than onelanguage. In some embodiments, a kit comprises one or more reagents foruse in a process utilizing one or more of the elements described herein.Reagents may be provided in any suitable container. For example, a kitmay provide one or more delivery or storage buffers. Reagents may beprovided in a form that is usable in a particular process, or in a formthat requires addition of one or more other components before use (e.g.in concentrate or lyophilized form). A buffer can be any buffer,including but not limited to a sodium carbonate buffer, a sodiumbicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, aHEPES buffer, and combinations thereof. In some embodiments, the bufferis alkaline. In some embodiments, the buffer has a pH from about 7 toabout 10. In some embodiments, the kit comprises one or more of thevectors, proteins and/or one or more of the polynucleotides describedherein. The kit may advantageously allow the provision of all elementsof the systems of the invention. Kits can involve vector(s) and/orparticle(s) and/or nanoparticle(s) containing or encoding RNA(s) for1-50 or more neoantigen mutations to be administered to an animal,mammal, primate, rodent, etc., with such a kit including instructionsfor administering to such a eukaryote, as well as instructions for usewith any of the methods of the present invention.

In one embodiment the kit contains at least one vial with an immunogeniccomposition or vaccine. In one embodiment kits may comprise ready to usecomponents that are mixed and ready to use. The ready to use immunogenicor vaccine composition may comprise separate vials containing differentpools of immunogenic compositions. The immunogenic compositions maycomprise one vial containing a viral vector or DNA plasmid and the othervial may comprise immunogenic protein. In another embodiment a kit maycontain an immunogenic composition or vaccine in a ready to bereconstituted form. The immunogenic or vaccine composition may be freezedried or lyophilized. The kit may comprise a separate vial with areconstitution buffer that can be added to the lyophilized compositionso that it is ready to administer. The buffer may advantageouslycomprise an adjuvant or emulsion according to the present invention. Inanother embodiment the kit may comprise single vials containing a doseof immunogenic composition. In another aspect multiple vials areincluded so that one vial is administered according to a treatmenttimeline. In a further embodiment the vials are labeled for their properadministration to a patient in need thereof. The immunogen may be in alyophilized form, a dried form or in aqueous solution as describedherein. The immunogen may be a live attenuated virus, protein, ornucleic acid as described herein.

In another embodiment the kit may comprise separate vials for animmunogenic composition for use in priming an immune response andanother immunogenic composition to be used for boosting. In oneembodiment the priming immunogenic composition could be DNA or a viralvector and the boosting immunogenic composition may be protein. Eithercomposition may be lyophilized or ready for administering.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined in the appended claims.

The present invention is further illustrated in the following Exampleswhich are given for illustration purposes only and are not intended tolimit the invention in any way.

EXAMPLES Example 1

Cancer Vaccine Testing Protocol

The herein-described compositions and methods may be tested on 15patients with high-risk melanoma (fully resected stages IIIB, IIIC andIVM1a,b) according to the general flow process shown in FIG. 2. Patientsmay receive a series of priming vaccinations with a mixture ofpersonalized tumor-specific peptides and poly-ICLC over a 4 week periodfollowed by two boosts during a maintenance phase. All vaccinations aresubcutaneously delivered. The vaccine or immunogenic composition isevaluated for safety, tolerability, immune response and clinical effectin patients and for feasibility of producing vaccine or immunogeniccomposition and successfully initiating vaccination within anappropriate time frame. The first cohort can consist of 5 patients, andafter safety is adequately demonstrated, an additional cohort of 10patients may be enrolled. Peripheral blood is extensively monitored forpeptide-specific T-cell responses and patients are followed for up totwo years to assess disease recurrence.

As described herein, there is a large body of evidence in both animalsand humans that mutated epitopes are effective in inducing an immuneresponse and that cases of spontaneous tumor regression or long termsurvival correlate with CD8+ T-cell responses to mutated epitopes(Buckwalter and Srivastava P K. “It is the antigen(s), stupid” and otherlessons from over a decade of vaccitherapy of human cancer. Seminars inimmunology 20:296-300 (2008); Karanikas et al, High frequency ofcytolytic T lymphocytes directed against a tumor-specific mutatedantigen detectable with HLA tetramers in the blood of a lung carcinomapatient with long survival. Cancer Res. 61:3718-3724 (2001); Lennerz etal, The response of autologous T cells to a human melanoma is dominatedby mutated neoantigens. Proc Natl Acad Sci USA. 102:16013 (2005)) andthat “immunoediting” can be tracked to alterations in expression ofdominant mutated antigens in mice and man (Matsushita et al, Cancerexome analysis reveals a T-cell-dependent mechanism of cancerimmunoediting Nature 482:400 (2012); DuPage et al, Expression oftumor-specific antigens underlies cancer immunoediting Nature 482:405(2012); and Sampson et al, Immunologic escape after prolongedprogression-free survival with epidermal growth factor receptor variantIII peptide vaccination in patients with newly diagnosed glioblastoma JClin Oncol. 28:4722-4729 (2010)).

Next-generation sequencing can now rapidly reveal the presence ofdiscrete mutations such as coding mutations in individual tumors, mostcommonly single amino acid changes (e.g., missense mutations) and lessfrequently novel stretches of amino acids generated by frame-shiftinsertions/deletions/gene fusions, read-through mutations in stopcodons, and translation of improperly spliced introns (e.g., neoORFs).NeoORFs are particularly valuable as immunogens because the entirety oftheir sequence is completely novel to the immune system and so areanalogous to a viral or bacterial foreign antigen. Thus, neoORFs: (1)are highly specific to the tumor (i.e. there is no expression in anynormal cells); (2) can bypass central tolerance, thereby increasing theprecursor frequency of neoantigen-specific CTLs. For example, the powerof utilizing analogous foreign sequences in a therapeutic anti-cancervaccine was recently demonstrated with peptides derived from humanpapilloma virus (HPV). ˜50% of the 19 patients with pre-neoplastic,viral-induced disease who received 3-4 vaccinations of a mix of HPVpeptides derived from the viral oncogenes E6 and E7 maintained acomplete response for ≥24 months (Kenter et a, Vaccination againstHPV-16 Oncoproteins for Vulvar Intraepithelial Neoplasia NEJM 361:1838(2009)).

Sequencing technology has revealed that each tumor contains multiple,patient-specific mutations that alter the protein coding content of agene. Such mutations create altered proteins, ranging from single aminoacid changes (caused by missense mutations) to addition of long regionsof novel amino acid sequence due to frame shifts, read-through oftermination codons or translation of intron regions (novel open readingframe mutations; neoORFs). These mutated proteins are valuable targetsfor the host's immune response to the tumor as, unlike native proteins,they are not subject to the immune-dampening effects of self-tolerance.Therefore, mutated proteins are more likely to be immunogenic and arealso more specific for the tumor cells compared to normal cells of thepatient.

Utilizing recently improved algorithms for predicting which missensemutations create strong binding peptides to the patient's cognate MHCmolecules, a set of peptides representative of optimal mutated epitopes(both neoORF and missense) for each patient is identified andprioritized and up to 20 or more peptides are prepared for immunization(Zhang et al, Machine learning competition in immunology—Prediction ofHLA class I binding peptides J Immunol Methods 374:1 (2011); Lundegaardet al Prediction of epitopes using neural network based methods JImmunol Methods 374:26 (2011)). Peptides ˜20-35 amino acids in length issynthesized because such “long” peptides undergo efficientinternalization, processing and cross-presentation in professionalantigen-presenting cells such as dendritic cells, and have been shown toinduce CTLs in humans (Melief and van der Burg, Immunotherapy ofestablished (pre) malignant disease by synthetic long peptide vaccinesNature Rev Cancer 8:351 (2008)).

In addition to a powerful and specific immunogen, an effective immuneresponse advantageously includes a strong adjuvant to activate theimmune system (Speiser and Romero, Molecularly defined vaccines forcancer immunotherapy, and protective T cell immunity Seminars in Immunol22:144 (2010)). For example, Toll-like receptors (TLRs) have emerged aspowerful sensors of microbial and viral pathogen “danger signals”,effectively inducing the innate immune system, and in turn, the adaptiveimmune system (Bhardwaj and Gnjatic, TLR AGONISTS: Are They GoodAdjuvants? Cancer J. 16:382-391 (2010)). Among the TLR agonists,poly-ICLC (a synthetic double-stranded RNA mimic) is one of the mostpotent activators of myeloid-derived dendritic cells. In a humanvolunteer study, poly-ICLC has been shown to be safe and to induce agene expression profile in peripheral blood cells comparable to thatinduced by one of the most potent live attenuated viral vaccines, theyellow fever vaccine YF-17D (Caskey et al, Synthetic double-stranded RNAinduces innate immune responses similar to a live viral vaccine inhumans J Exp Med 208:2357 (2011)). Hiltonol®, a GMP preparation ofpoly-ICLC prepared by Oncovir, Inc, is utilized as the adjuvant.

Example 2

Target Patient Population

Patients with stage IIIB, IIIC and IVM1a,b, melanoma have a significantrisk of disease recurrence and death, even with complete surgicalresection of disease (Balch et al, Final Version of 2009 AJCC MelanomaStaging and Classification J Clin Oncol 27:6199-6206 (2009)). Anavailable systemic adjuvant therapy for this patient population isinterferon-α (IFNα) which provides a measurable but marginal benefit andis associated with significant, frequently dose-limiting toxicity(Kirkwood et al, Interferon alfa-2b Adjuvant Therapy of High-RiskResected Cutaneous Melanoma: The Eastern Cooperative Oncology GroupTrial EST 1684 J Clin Oncol 14:7-17 (1996); Kirkwood et al, High- andLow-dose Interferon Alpha-2b in High-Risk Melanoma: First Analysis ofIntergroup Trial E1690/59111/C9190 J Clin Oncol 18:2444-2458 (2000)).These patients are not immuno-compromised by previous cancer-directedtherapy or by active cancer and thus represent an excellent patientpopulation in which to assess the safety and immunological impact of thevaccine. Finally, current standard of care for these patients does notmandate any treatment following surgery, thus allowing for the 8-10 weekwindow for vaccine preparation.

The target population is cutaneous melanoma patients with clinicallydetectable, histologically confirmed nodal (local or distant) or intransit metastasis, who have been fully resected and are free of disease(most of stage IIIB (because of the need to have adequate tumor tissuefor sequencing and cell line development, patients with ulceratedprimary tumor but micrometastatic lymph nodes (T1-4b, N1a or N2a) isexcluded), all of stage IIIC, and stage IVM1a, b). These may be patientsat first diagnosis or at disease recurrence after previous diagnosis ofan earlier stage melanoma.

Tumor harvest: Patients can undergo complete resection of their primarymelanoma (if not already removed) and all regional metastatic diseasewith the intent of rendering them free of melanoma. After adequate tumorfor pathological assessment has been harvested, remaining tumor tissueis placed in sterile media in a sterile container and prepared fordisaggregation. Portions of the tumor tissue is used for whole-exome andtranscriptome sequencing and cell line generation and any remainingtumor is frozen.

Normal tissue harvest: A normal tissue sample (blood or sputum sample)is taken for whole exome sequencing.

Patients with clinically evident locoregional metastatic disease orfully resectable distant nodal, cutaneous or lung metastatic disease(but absence of unresectable distant or visceral metastatic disease) isidentified and enrolled on the study. Entry of patients prior to surgeryis necessary in order to acquire fresh tumor tissue for melanoma cellline development (to generate target cells for in vitro cytotoxicityassays as part of the immune monitoring plan).

Example 3

Dose and Schedule

For patients who have met all pre-treatment criteria, vaccineadministration can commence as soon as possible after the study drug hasarrived and has met incoming specifications. For each patient, there isfour separate study drugs, each containing 5 of 20 patient-specificpeptides. Immunizations may generally proceed according to the scheduleshown in FIG. 3.

Patients are treated in an outpatient clinic. Immunization on eachtreatment day can consist of four 1 ml subcutaneous injections, eachinto a separate extremity in order to target different regions of thelymphatic system to reduce antigenic competition. If the patient hasundergone complete axillary or inguinal lymph node dissection, vaccinesare administered into the right or left midriff as an alternative. Eachinjection can consist of 1 of the 4 study drugs for that patient and thesame study drug is injected into the same extremity for each cycle. Thecomposition of each 1 ml injection is:

0.75 ml study drug containing 300 μg each of 5 patient-specific peptides

0.25 ml (0.5 mg) of 2 mg/ml poly-ICLC (Hiltonol®)

During the induction/priming phase, patients are immunized on days 1, 4,8, 15 and 22. In the maintenance phase, patients can receive boosterdoses at weeks 12 and 24.

Blood samples may be obtained at multiple time points: pre- (baseline;two samples on different days); day 15 during priming vaccination; fourweeks after the induction/priming vaccination (week 8); pre- (week 12)and post- (week 16) first boost; pre- (week 24) and post-(week 28)second boost 50-150 ml blood is collected for each sample (except week16). The primary immunological endpoint is at week 16, and hencepatients can undergo leukapheresis (unless otherwise indicated based onpatient and physician assessment).

Example 4

Immune Monitoring

The immunization strategy is a “prime-boost” approach, involving aninitial series of closely spaced immunizations to induce an immuneresponse followed by a period of rest to allow memory T-cells to beestablished. This is followed by a booster immunization, and the T-cellresponse 4 weeks after this boost is expected to generate the strongestresponse and is the primary immunological endpoint. Global immunologicalresponse is initially monitored using peripheral blood mononuclear cellsfrom this time point in an 18 hr ex vivo ELISPOT assay, stimulating witha pool of overlapping 15mer peptides (11 aa overlap) comprising all theimmunizing epitopes. Pre-vaccination samples are evaluated to establishthe baseline response to this peptide pool. As warranted, additionalPBMC samples are evaluated to examine the kinetics of the immuneresponse to the total peptide mix. For patients demonstrating responsessignificantly above baseline, the pool of all 15mers are de-convolutedto determine which particular immunizing peptide(s) were immunogenic. Inaddition, a number of additional assays are conducted on a case-by-casebasis for appropriate samples:

-   -   The entire 15mer pool or sub-pools are used as stimulating        peptides for intracellular cytokine staining assays to identify        and quantify antigen-specific CD4+, CD8+, central memory and        effector memory populations    -   Similarly, these pools are used to evaluate the pattern of        cytokines secreted by these cells to determine the TH1 vs TH2        phenotype    -   Extracellular cytokine staining and flow cytometry of        unstimulated cells are used to quantify Treg and myeloid-derived        suppressor cells (MDSC).    -   If a melanoma cell line is successfully established from a        responding patient and the activating epitope can be identified,        T-cell cytotoxicity assays are conducted using the mutant and        corresponding wild type peptide    -   PBMC from the primary immunological endpoint is evaluated for        “epitope spreading” by using known melanoma tumor associated        antigens as stimulants and by using several additional        identified mutated epitopes that were not selected to be among        the immunogens, as shown in FIG. 4.

Immuno-histochemistry of the tumor sample is conducted to quantify CD4+,CD8+, MDSC, and Treg infiltrating populations.

Example 5

Neoantigen Preparation

Following surgical resection of the tumor, a portion of the tumor tissueand a blood sample is transferred immediately to the facility where itis assigned a unique identification code for further tracking. The tumortissue is disaggregated with collagenase and separate portions arefrozen for nucleic acid (DNA and RNA) extraction. The blood sample isimmediately transferred to a facility for nucleic acid extraction. DNAand/or RNA extracted from the tumor tissue is used for whole-exomesequencing (e.g., by using the Illumina HiSeq platform) and to determineHLA typing information. It is contemplated within the scope of theinvention that missense or neoORF neoantigenic peptides may be directlyidentified by protein-based techniques (e.g., mass spectrometry).

Bioinformatics analysis are conducted as follows. Sequence analysis ofthe Exome and RNA—SEQ fast Q files leverage existing bioinformaticpipelines that have been used and validated extensively in large-scaleprojects such as the TCGA for many patient samples (e.g., Chapman et al,2011, Stransky et al, 2011, Berger et al, 2012). There are twosequential categories of analyses: data processing and cancer genomeanalysis.

Data processing pipeline: The Picard data processing pipeline(picard.sourceforge.net/) was developed by the Sequencing Platform. Rawdata extracted from (e.g., Illumina) sequencers for each tumor andnormal sample is subjected to the following processes using variousmodules in the Picard pipeline:

-   -   (i) Data conversion: Raw Illumina data is converted to the        standard BAM format and basic QC metrics pertaining to the        distribution of bases exceeding different quality thresholds are        generated.    -   (ii) Alignment: The Burrows-Wheeler Alignment Tool (BWA) is used        to align read pairs to the human genome (hg19).    -   (iii) Mark Duplicates: PCR and optical duplicates are identified        based on read pair mapping positions and marked in the final BAM        file.    -   (iv) Indel Realignment: Reads that align to known insertion and        deletion polymorphic sites in the genome is examined and those        sites where the log odds (LOD) score for improvement upon        realignment is at least 0.4 is corrected.    -   (v) Quality Recalibration: Original base quality scores reported        by the Illumina pipeline is recalibrated based on the        read-cycle, the lane, the flow cell tile, the base in question        and the preceding base. The recalibration assumes that all        mismatches in non-dbSNP positions are due to errors which enable        recalibration of the probability of error in each category of        interest as the fraction of mismatches amongst the total number        of observations.    -   (vi) Quality Control: The final BAM file is processed to        generate extensive QC metrics including read quality by cycle,        distribution of quality scores, summary of alignment and the        insert size distribution. Data that fails quality QC is        blacklisted.    -   (vii) Identity Verification: Orthogonally collected sample        genotype data at ˜100 known SNP positions are checked against        the sequence data to confirm the identity of the sample. A LOD        score of ≥10 is used as a threshold for confirmation of        identity. Data that fails identity QC is blacklisted.    -   (viii) Data Aggregation: All data from the same sample is merged        and the mark duplicates step is repeated. Novel target regions        containing putative short insertions and deletion regions are        identified and the indel realignment step is performed at these        loci.    -   (ix) Local realignment around putative indels in aggregated        data: Novel target regions containing putative short insertions        and deletions are identified and a local realignment step is        performed at these loci (e.g., using the GATK        RealignerTargetCreator and IndelRealigner modules) to ensure        consistency and correctness of indel calls.    -   (x) Quality Control on Aggregated Data: QC metrics such as        alignment summary and insert size distribution is recomputed.        Additionally a set of metrics that evaluate the rate of        oxidative damage in the early steps of the library constructions        process caused by acoustic shearing of DNA in the presence of        reactive contaminants from the extraction process are generated.

The output of Picard is a bam file (Li et al, 2009) (see, e.g.,http://samtools.sourceforge.net/SAM1.pdf) that stores the basesequences, quality scores, and alignment details for all reads for thegiven sample.

Cancer Mutation Detection Pipeline: Tumor and matched normal bam filesfrom the Picard pipeline is analyzed as described herein:

-   -   1. Quality Control        -   (i). The Capseg program is applied to tumor and matched            normal exome samples to get the copy number profiles. The            CopyNumberQC tool can then be used to manually inspect the            generated profiles and assess tumor/normal sample mix-ups.            Normal samples that have noisy profiles as well as cases            where the tumor sample has lower copy number variation than            the corresponding normal is flagged and tracked through the            data generation and analysis pipelines to check for mix-ups.        -   (ii). Tumor purity and ploidy is estimated by the ABSOLUTE            tool 15 based on Capseg-generated copy number profiles. Very            noisy profiles might result from sequencing of highly            degraded samples. No tumor purity and ploidy estimates would            be possible in such cases and the corresponding sample is            flagged.        -   (iii). ContEst (Cibulskis et al, 2011) is used to determine            the level of cross-sample contamination in samples. Samples            with greater than 4% contamination is discarded.    -   2. Identification of somatic single nucleotide variations        (SSNVs)        -   Somatic base pair substitutions are identified by analyzing            tumor and matched normal bams from a patient using a            Bayesian statistical framework called muTect (Cibulskis et            al, 2013). In the preprocessing step, reads with a            preponderance of low quality bases or mismatches to the            genome are filtered out. Mutect then computes two log-odds            (LOD) scores which encapsulate confidence in presence and            absence of the variant in the tumor and normal samples            respectively. In the post-processing stage candidate            mutations are filtered by six filters to account for            artifacts of capture, sequencing and alignment:            -   (i) Proximal gap: removes false positives that arise due                to the presence of misaligned indels in the vicinity of                the event. Samples with ≥3 reads with insertions or                deletions in a 11-bp window around the candidate                mutation are rejected.            -   (ii) Poor mapping: discards false positives that arise                by virtue of ambiguous placement of reads in the genome.                Rejects candidates if ≥50% reads in tumor and normal                samples have mapping quality zero or if there are no                reads harboring the mutant allele with mapping quality                ≥20.            -   (iii) Trialleleic sites: discards sites that are                heterozygous in the normal since these have a tendency                to generate many false positives.            -   (iv) Strand bias: removes false positives caused by                context-specific sequencing errors where a large                fraction of reads harboring the mutation have the same                orientation. Rejects candidates where the                strand-specific LOD is <2 where the sensitivity to pass                that threshold is ≥90%.            -   (v) Clustered position: rejects false positives due to                alignment errors characterized by the alternative allele                occurring at a fixed distance from the start or end of                the read alignment. Rejects if the median distance from                the start and end of the reads are ≤10 which implies                that the mutation is at the start or end of the                alignment, or if the median absolute deviation of the                distances are ≤3 which implies that the mutations are                clustered.            -   (vi) Observed in control: discards false positives in                the tumor where there is evidence of occurrence of the                alternate allele in the normal sample beyond what is                expected by random sequencing errors. Rejects if there                are ≥2 reads containing the alternate allele in the                normal sample or if they are in ≥3% of the reads, and if                the sum of their quality scores are >20.        -   In addition to these 6 filters, candidates are compared            against a panel of normal samples and those that are found            to be present as germline variants in two or more normal            samples are rejected. The final set of mutations can then be            annotated with the Oncotator tool by several fields            including genomic region, codon, cDNA and protein changes.    -   3. Identification of somatic small insertions and deletions        -   The local realignment output described herein (see “Local            realignment around putative indels in aggregated data”,            supra) is used to predict candidate somatic and germline            indels based on assessment of reads supporting the variant            exclusively in tumor or both in tumor and normal bams            respectively. Further filtering based on number and            distribution of mismatches and base quality scores are done            (McKenna et al, 2010, DePristo et al, 2011). All indels are            manually inspected using the Integrated Genomics Viewer            (Robinson et al, 2011) (www.broadinstitute.org/igv) to            ensure high-fidelity calls.    -   4. Gene fusion detection        -   The first step in the gene fusion detection pipeline is            alignment of tumor RNA-Seq reads to a library of known gene            sequences following by mapping of this alignment to genomic            coordinates. The genomic mapping helps collapse multiple            read pairs that map to different transcript variants that            share exons to common genomic locations. The DNA aligned bam            file is queried for read pairs where the two mates map to            two different coding regions that are either on different            chromosomes or at least 1 MB apart if on the same            chromosome. It can also be required that the pair ends            aligned in their respective genes be in the direction            consistent with coding-->coding 5′->3′ direction of the            (putative) fusion mRNA transcript. A list of gene pairs            where there are at least two such ‘chimeric’ read pairs are            enumerated as the initial putative event list subject to            further refinement. Next, all unaligned reads are extracted            from the original bam file, with the additional constraint            that their mates were originally aligned and map into one of            the genes in the gene pairs obtained as described herein. An            attempt can then be made to align all such originally            unaligned reads to the custom “reference” built of all            possible exon-exon junctions (full length,            boundary-to-boundary, in coding 5′->3′ direction) between            the discovered gene pairs. If one such originally unaligned            read maps (uniquely) onto a junction between an exon of gene            X and an exon of gene Y, and its mate was indeed mapped to            one of the genes X or Y, then such a read is marked as a            “fusion” read. Gene fusion events are called in cases where            there is at least one fusion read in correct relative            orientation to its mate, without excessive number of            mismatches around the exon:exon junction and with a coverage            of at least 10 bp in either gene. Gene fusions between            highly homologous genes (ex. HLA family) are likely spurious            and is filtered out.    -   5. Estimation of clonality        -   Bioinformatic analysis may be used to estimate clonality of            mutations. For example, the ABSOLUTE algorithm (Carter et            al, 2012, Landau et al, 2013) may be used to estimate tumor            purity, ploidy, absolute copy numbers and clonality of            mutations. Probability density distributions of allelic            fractions of each mutation is generated followed by            conversion to cancer cell fractions (CCFs) of the mutations.            Mutations are classified as clonal or subclonal based on            whether the posterior probability of their CCF exceeds 0.95            is greater or lesser than 0.5 respectively.    -   6. Quantification of expression        -   The TopHat suite (Langmead et al, 2009) is used to align            RNA-Seq reads for the tumor and matched normal bams to the            hg19 genome. The quality of RNA-Seq data is assessed by the            RNA-SeQC (DeLuca et al., 2012) package. The RSEM tool (Li et            al., 2011) can then be used to estimate gene and isoform            expression levels. The generated reads per kilobase per            million and tau estimates are used to prioritize neoantigens            identified in each patient as described elsewhere.    -   7. Validation of mutations in RNA-Seq    -   8. Confirmation of the somatic mutations identified by analysis        of whole exome data as described herein (including single        nucleotide variations, small insertions and deletions and gene        fusions) are assessed by examining the corresponding RNA-Seq        tumor BAM file of the patient. For each variant locus, a power        calculation based on the beta-binomial distribution is performed        to ensure that there is at least 95% power to detect it in the        RNA-Seq data. A capture identified mutation is considered        validated if there are at least 2 reads harboring the mutation        for adequately powered sites.

Selection of Tumor-Specific Mutation-Containing Epitopes: All missensemutations and neoORFs are analyzed for the presence ofmutation-containing epitopes using the neural-network based algorithmnetMHC, provided and maintained by the Center for Biological SequenceAnalysis, Technical University of Denmark, Netherlands. This family ofalgorithms were rated the top epitope prediction algorithms based on acompetition recently completed among a series of related approaches(ref). The algorithms were trained using an artificial neural networkbased approach on 69 different human HLA A and B alleles covering 99% ofthe HLA-A alleles and 87% of the HLA-B alleles found in the Caucasianpopulation, the major ethnic group in the target patient population inthe local area. The most up-do-date version is utilized (v2.4).

The accuracy of the algorithms were evaluated by conducting predictionsfrom mutations found in CLL patients for whom the HLA allotypes wereknown. The included allotypes were A0101, A0201, A0310, All01, A2402,A6801, B0702, B0801, B1501. Predictions were made for all 9mer and 10mer peptides spanning each mutation using netMHCpan in mid-2011. Basedon these predictions, seventy-four (74) 9mer peptides and sixty-three(63) 10mer peptides, most with predicted affinities below 500 nM, weresynthesized and the binding affinity was measured using a competitivebinding assay (Sette).

The predictions for these peptides were repeated in March 2013 usingeach of the most up to date versions of the netMHC servers (netMHCpan,netMHC and netMHCcons). These three algorithms were the top ratedalgorithms among a group of 20 used in a competition in 2012 (Zhang etal). The observed binding affinities were then evaluated with respect toeach of the new predictions. For each set of predicted and observedvalues, the % of correct predictions for each range is given, as well asthe number of samples. The definition for each range is as follows:

-   -   0-150: Predicted to have an affinity equal to or lower than 150        nM and measured to have an affinity equal to or lower than 150        nM.    -   0-150*: Predicted to have an affinity equal to or lower than 150        nM and measured to have an affinity equal to or lower than 500        nM.    -   151-500 nM: Predicted to have an affinity greater than 150 nM        but equal to or lower than 500 nM and measured to have an        affinity equal to or below 500 nM.    -   FN (>500 nM): False Negatives—Predicted to have an affinity        greater than 500 nM but measured to have an affinity equal to or        below 500 nM.

For 9mer peptides (Table 1), there was little difference between thealgorithms, with the slightly higher value for the 151-500 nM range fornetMHC cons not judged to be significant because of the low number ofsamples.

TABLE 1 Range (nM) 9mer PAN 9mer netMHC 9mer CONS 0-150 76% 78% 76% (33)(37) (34) 0-150* 91% 89% 88% (33) (37) (34) 151-500 50% 50% 62% (28)(14) (13) FN (>500) 38% 39% 41% (13) (23) (27)

For 10mer peptides (Table 2), again there was little difference betweenthe algorithms except that netMHC produced significantly more falsepositives than netMHCpan or netMMHCcons. However, the precision of the10mer predictions are slightly lower in the 0-150 nM and 0-150* nMranges and significantly lower in the 151-500 nM range, compared to the9mers.

TABLE 2 Range (nM) 10mer PAN 10mer netMHC 10mer CONS 0-150 53% 50% 59%(19) (16) (17) 0-150* 68% 69% 76% (19) (16) (17) 151-500 35% 42% 35%(26) (12) (23) FN (>500) 11% 23% 13% (18) (35) (23)

For 10mers, only predictions in the 0-150 nM range is utilized due tothe lower than 50% precision for binders in the 151-500 nM range.

The number of samples for any individual HLA allele was too small todraw any conclusions regarding accuracy of the prediction algorithm fordifferent alleles. Data from the largest available subset (0-150* nM;9mer) is shown in Table 3 as an example.

TABLE 3 Fraction Allele correct A0101 2/2 A0201  9/11 A0301 5/5 A11014/4 A2402 0/0 A6801 3/4 B0702 4/4 B0801 1/2 B1501 2/2

Only predictions for HLA A and B alleles are utilized as there is littleavailable data on which to judge accuracy of predictions for HLA Calleles (Zhang et al).

An evaluation of melanoma sequence information and peptide bindingpredictions was conducted using information from the TCGA database.Information for 220 melanomas from different patients revealed that onaverage there were approximately 450 missense and 5 neoORFs per patient.20 patients were selected at random and the predicted binding affinitieswere calculated for all the missense and neoORF mutations using netMHC(Lundegaard et al Prediction of epitopes using neural network basedmethods J Immunol Methods 374:26 (2011)). As the HLA allotypes wereunknown for these patients, the number of predicted binding peptides perallotype were adjusted based on the frequency of that allotype (BoneMarrow Registry dataset for the expected affected dominant population inthe geographic area [Caucasian for melanoma]) to generate a predictednumber of actionable mutant epitopes per patient. For each of thesemutant epitopes (MUT), the corresponding native (NAT) epitope bindingwas also predicted.

Utilizing the Prioritization Described Herein:

-   -   90% (18 of 20) of patients were predicted to have at least 20        peptides appropriate for vaccination;    -   For nearly a quarter of the patients, neoORF peptides could        constitute half to all of the 20 peptides;    -   For just over half of the patients, only peptides in categories        1 and 2 would be used;    -   For 80% of the patients, only peptides in categories 1, 2, and 3        would be utilized.

Thus, there is a sufficient number of mutations in melanoma to expect ahigh proportion of patients to generate an adequate number ofimmunogenic peptides.

Example 6

Peptide Production and Formulation

GMP neoantigenic peptides for immunization is prepared by chemicalsynthesis Merrifield R B: Solid phase peptide synthesis. I. Thesynthesis of a tetrapeptide. J. Am. Chem. Soc. 85:2149-54, 1963) inaccordance with FDA regulations. Three development runs have beenconducted of 20 ˜20-30mer peptides each. Each run was conducted in thesame facility and utilized the same equipment as is used for the GMPruns, utilizing draft GMP batch records. Each run successfullyproduced >50 mg of each peptide, which were tested by all currentlyplanned release tests (e.g., Appearance, Identify by MS, Purity byRP-HPLC, Content by Elemental Nitrogen, and TFA content by RP-HPLC) andmet the targeted specification where appropriate. The products were alsoproduced within the timeframe anticipated for this part of the process(approximately 4 weeks). The lyophilized bulk peptides were placed on along term stability study and is evaluated at various time points up to12 months.

Material from these runs has been used to test the planned dissolutionand mixing approach. Briefly, each peptide is dissolved at highconcentration (50 mg/ml) in 100% DMSO and diluted to 2 mg/ml in anaqueous solvent. Initially, it was anticipated that PBS would be used asa diluent, however, a salting out of a small number of peptides caused avisible cloudiness. D5W (5% dextrose in water) was shown to be much moreeffective; 37 of 40 peptides were successfully diluted to a clearsolution. 10% sucrose or 10% Trehalose in water also is effective. Theformulation containing 10% sucrose or 10% trehalose is lyophilizableunlike the formulation containing 5% Dextrose. The only problematicpeptides are very hydrophobic peptides.

Table 4 shows the results of solubility evaluations of 60 potentialneoantigen peptides, sorted based on the calculated fraction ofhydrophobic amino acids. As shown, almost all peptides with ahydrophobic fraction lower than 0.4 are soluble in DMSO/D5W, but anumber of peptides with a hydrophobic fraction greater than or equal to0.4 were not soluble in DMSO/D5W (indicated by bold font in the columnlabeled “Solubility in DMSO/D5W”). A number of these can be solubilizedby addition of succinate (indicated by underlining in the column“Solubility in DMSO/D5W/Succinate”). 3 of 4 of these peptides hadhydrophobic fractions between 0.4 and 0.43. Four peptides became lesssoluble upon addition of succinate; 3 of 4 of these peptides had ahydrophobic fraction greater than or equal to 0.45.

TABLE 4  pHof pHof pep- pep- tides tides in in DMSO/   Solu- DMSO/ D5W/pHof bility  D5W/ 5nnM Solu- pep- in 5mM Suc- Solu- bility tides DMSO/S. cinate Approx. bility In in D5W/ Suc- and Hydro- Iso- in DMSO/ DMSO/Suc- cinate Hil- phobi- Hydro- electric ID SEQUENCES DMSO D5W D5W cinatespike tonol city phillic Point CS6715 PPYPYSSPSLVLPT Y 4.11 0.17 0.107.86 EPHTPKSLQQPG LPS CS6722 NPEKYKAKSRSPG 0.18 0.27 9.45 SPVVEGTGSPPKWQIGEQEF CS6725 GTYLQGTASALS Y 3.95 0.18 0.12 7.03 QSQERPPSVNRVPPSSPSSQE CS7416 AESAQRQGPNG Y 3.91 Y 6.31 6.54 0.20 0.20 3.73 GGEQSANEFCS6710 EPDQEAVQSSTY Y 3.65 0.21 0.31 4.71 KDCNTLHLPTERF SPVR CS6712LKDSNSWPPSNK 0.21 0.31 7.95 RGFDTEDAHKSN ATPVP CS6781 GASRRSSASQGA Y0.21 0.21 11.26 GSLGLSEEKTLRS GGGP CS6718 KKEKAEKLEKERQ Y 0.21 0.4510.31 RHISKPLLGGPFSL TTHTGE CS6720 SPTEPSTKLPGFD Y 0.21 0.30 9.48SCGNTEIAERKIK RIYGGFK CS6723 ECGKAFTRGSQL Y 3.68 0.21 0.33 6.14TQHQGIHISEKSF EYKECGID CS6708 SHVEKAHITAESA Y 0.24 0.28 5.25 QRQGPNGGGEQSANEF CS6721 PIERVKKNLLKKE Y 0.24 0.39 9.33 YNVSDDSMKLG GNNTSEKAD CS6916HKSIGQPKLSTHP Y 0.25 0.22 10.64 FLCPKPQKMNTS LGQHLTL CS7417 AESAQRQGPLGGY 3.82 Y 6.28 6.5 0.25 0.20 3.73 GEQSANEF CS6717 KPKKVAGAATPK Y 4.650.27 0.39 12.18 KSIKRTPKKVKKP ATAAGTKK CS6719 SKLPYPVAKSGKR Y 3.94 0.270.24 11.1 ALARGPAPTEKTP HSGAQLG CS6925 EQGPWQSEGQT Y 0.28 0.14 6.14WRAAGGRVPVP CPAAGPG CS6915 SGARIGAPPPHA Y 0.30 0.17 8.02 TATSSSSFMPGTWGREDL CS6919 KLAWRGRISSSG Y 4.38 Y 6.74 6.99 0.30 0.13 11.38CPSMTSPPSPMF GMTLHT CS6726 DSAVDKGHPNRS Y 0.30 0.18 10.26 ALSLTPGLRIGPSGIPQAGLG CS7409 LLTDRNTSGTTFT Y 3.86 Y 6.32 6.62 0.31 0.15 3.59LLGVSDYPELQVP CS6709 LTDLPGRIRVAPQ X NT 0.31 0.21 3.91 QNDLDSPQQISIS NAECS7414 KGASLDAGWGS Y 3.81 Y 6.71 6.99 0.31 0.21 12.5 PRWTTTRMTSASAGRSTRA CS6917 FRLIWRSVKNGK Y 0.31 0.25 10.67 SSREQELSWNCS HQVPSLGACS6938 GKSRGQQAQDR Y 0.33 0.30 12.31 ARHAAGAAPARP LGALREQ CS7408LLTDRNTSGTTFT Y 3.89 Y 6.31 6.75 0.33 0.12 3.59 LLGVSDYPELQVP IPQAGLGCS6711 RGLHSQGLGRGR Y 3.82 0.34 0.28 10.92 IAMAQTAGVLRS LEQEE CS6716PQLAGGGGSGAP Y 0.34 0.07 5.08 GEHPLLPGGAPL PAGLF CS6926 TWAGHVSTALAR Y0.34 0.10 7.05 PLGAPWAEPGSC GPGTN CS7431 KKNITNLSRLVVR Y 3.8 Y 6.45 6.690.35 0.30 10.29 PDTDAVY CS7432 WDGPPENDMLL Y 3.72 Y 6.22 6.45 0.35 0.253.43 KEICGSLIP CS6930 LAASGLHGSAWL Y 0.35 0.16 8.17 VPGEQPVSGPHH GKQPAGVCS6729 PIQVFYTKQPQN Y 3.87 0.36 0.15 6.15 DYLHVALVSVFQI HQEAPSSQ CS6931VAGLAASGLHGS Y 3.80 Y 6.42 6.66 0.37 0.17 8.17 AWLVPGEQPVS GPHHGKQCS6934 SKRGVGAKTLLLP Y 3.86 Y 6.57 6.79 0.38 0.24 10.67 DPFLFWPCLEGTRRSL CS6936 SYKKLPLLIFPSHR Y 0.38 0.24 11.48 RAPLLSATGDRGF SV CS6914GLLSDGSGLGQIT Y 0.40 0.17 4.4 WASAEHLQRPG AGAELA CS6932 DLCICPRSHRGAF Y0.40 0.23 6.9 QLLPSALLVRVLE GSDS CS6935 DASDFLPDTQLFP N Y 0.40 0.23 3.2HFTELLLPLDPLE GSSV CS6943 DMAWRRNSRLY Y 0.40 0.27 9.79 WLIKMVEQWQEQHLPSLSS CS7428 LSVPFTCGVNFG N n/a Y n/a n/a 0.40 0.20 2.75 DSIEDLEICS7430 PLMQTELHQLVP Y 3.95 Y 6.23 6.37 0.40 0.30 3.35 EADPEEMA CS6918EDLHLLSVPCPSY Y 0.41 0.25 9.67 KKLPLLIFPSHRRA PLLSA CS6941 AHRQGEKQHLLPY 3.92 Y 6.49 6.78 0.41 0.31 12.5 VFSRLALRLPWR HSVQL CS7410ALSLTPGLRIGPS Y 3.99 Y 6.46 6.88 0.42 0.18 10.26 GLFLVFLAESAVD KGHPNRSCS7411 DSAVDKGHPNRS Y 3.87 Y 6.53 6.94 0.42 0.18 10.26 ALSLTPGLRIGPSGLFLVFLA CS7412 LRVFIGNIAVNHA Y 4.24 N 6.61 6.96 0.42 0.09 12.49PVSLRPGLGLPPG APPGTVP CS7438 LPVFIGNIAVNHA Y 4.24 Y 6.78 6.96 0.42 0.0611.18 PVSLRPGLGLPPG APPGTVP CS6942 VSWGKKVQPIDS N Y 0.43 0.37 3.68ILADWNEDIEAFE MMEKD CS7415 GTKALQLHSIAGR Y 3.91 Y 6.61 6.81 0.43 0.2010.26 WPRMEPWVVES MSLGVP CS6937 SGQPAPEETVLFL Y 3.87 N 6.51 6.76 0.450.21 10.98 GLLHGLLLILRRLR GG CS7418 YLLPKTAVVLRCP Y 3.98 Y 6.76 6.960.45 0.25 11.48 ALRVRKP CS7420 IGALNPKRAAFFA Y 3.84 N 6.38 6.56 0.450.30 5.38 EHYESWE CS7425 SYDSVIRELLQKP X Y 3.78 N 6.44 6.65 0.45 0.259.79 NVRVVVL CS7427 VEQGHVRVGPD Y 3.72 Y 6.34 6.52 0.45 0.25 6.15VVTHPAFLV CS6927 APALGPGAASVA Y 0.45 0.13 8.99 SRCGLDPALAPG GSHMLRACS6783 LLTDRNTSGTTFT N 3.96 Y 0.45 0.12 3.59 LLGVSDYPELQVP LFLVFLACS6933 EEGLLPEVFGA Y 0.45 0.21 7.05 GVPLALCPAVP SAAKPHRPRVL CS7413VQLSIQDVIRR Y 3.9 Y 6.73 7.02 0.47 0.20 12.68 ARLSTVPTAQR VALRSGWICS6730 LPVFIGNIAVN Y 4.20 0.48 0.06 11.18 HAPVSLRPGLG LPPGAPPLVVP

The predicted biochemical properties of planned immunizing peptides areevaluated and synthesis plans may be altered accordingly (using ashorter peptide, shifting the region to be synthesized in the N- orC-terminal direction around the predicted epitope, or potentiallyutilizing an alternate peptide) in order to limit the number of peptideswith a high hydrophobic fraction.

Ten separate peptides in DMSO/D5W were subjected to two freeze/thawcycles and showed full recovery. Two individual peptides were dissolvedin DMSO/D5W and placed on stability at two temperatures (−20° C. and−80° C.). These peptides were evaluated (RP-HPLC and pH and visualinspection) for up to 24 weeks. Both peptides are stable for up to 24weeks; the percent impurities detected by the RP-HPLC assay did notchange significantly for either peptide when stored at either −20° C. or−80° C. Any small changes appear to be due to assay variability as notrends were noted to be evaluated.

As shown in FIG. 5, the design of the dosage form process are to prepare4 pools of patient-specific peptides consisting of 5 peptides each. ARP-HPLC assay has been prepared and qualified to evaluate these peptidemixes. This assay achieves good resolution of multiple peptides within asingle mix and can also be used to quantitate individual peptides.

Membrane filtration (0.2 μm pore size) is used to reduce bioburden andconduct final filter sterilization. Four different appropriately sizedfilter types were initially evaluated and the Pall, PES filter (#4612)was selected. To date, 4 different mixtures of 5 different peptides eachhave been prepared and individually filtered sequentially through twoPES filters. Recovery of each individual peptide was evaluated utilizingthe RP-HPLC assay. For 18 of the 20 peptides, the recovery after twofiltrations was >90%. For two highly hydrophobic peptides, the recoverywas below 60% when evaluated at small scale but were nearly fullyrecovered (87 and 97%) at scale. As stated herein, approaches areundertaken to limit the hydrophobic nature of the sequences selected.

A peptide pool (Pool 4) consisting of five peptides was prepared bydissolution in DMSO, dilution with D5W/Succinate (5 mM) to 2 mg/ml andpooling to a final peptide concentration of 400 μg per ml and a finalDMSO concentration of 4%. After preparation, peptides were filtered witha 25 mm Pall PES filter (Cat #4612) and dispensed into Nunc Cryo vials(#375418) in one ml aliquots. Samples were analyzed at time zero and at2 and 4 weeks to date. Additional samples are analyzed at 8 and 24weeks. At −80° C., no significant change in the HPLC profiles orimpurity profile of the peptide Pool 4 was observed at the four-weektime point. Through the 4 week time point, visual observation and pH forthe peptide pool did not change.

Example 7

Peptide Synthesis

GMP peptides are synthesized by standard solid phase synthetic peptidechemistry (e.g., using CS 536 XT peptide synthesizers) and purified byRP-HPLC. Each individual peptide is analyzed by a variety of qualifiedassays to assess appearance (visual), purity (RP-HPLC), identity (bymass spectrometry), quantity (elemental nitrogen), and trifluoroacetatecounterion (RP-HPLC) and released.

The personalized neoantigen peptides may be comprised of up to 20distinct peptides unique to each patient. Each peptide may be a linearpolymer of ˜20-˜30 L-amino acids joined by standard peptide bonds. Theamino terminus may be a primary amine (NH2-) and the carboxy terminus isa carbonyl group (—COOH). The standard 20 amino acids commonly found inmammalian cells are utilized (alanine, arginine, asparagine, asparticacid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, valine). The molecular weight of eachpeptide varies based on its length and sequence and is calculated foreach peptide.

Fmoc (9-fluorenylmethoyloxycarbnyl)-N-terminal protected amino acids areutilized for all synthesis reactions. The side chains of the amino acidsare protected by 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl(Pbf), triphenylmethyl (Trt), t-butyloxycarbonyl (Boc) or t-butyl ether(tBu) group as appropriate. All bulk amino acids are dissolved indimethylformamide (DMF). Condensation utilizes the following twocatalyst combinations in separate reactions:

-   -   Diisopylcarbodiimide/1-Hydroxybenzotriazole (DIC/HOBT)    -   Diisoproplyethylamine/2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium        hexafluorophosphate (DIEA/HBTU)

Each amino acid is coupled twice in order to ensure high level ofincorporation. The first coupling utilizes DIC/HOBT for 2-6 hours andthe second coupling utilizes DIEA/HBTU for 1-2 hours. Each of the twocouplings are monitored by UV absorbance and the resin is washedextensively with DMF in between coupling cycles to improve efficiency.After two cycles of coupling, calculated coupling efficiency must be atleast 95% to continue to the next cycle. Further synthesis of anypeptides that do not meet that minimal coupling efficiency is stopped.

After all amino acids have been coupled, the resin is washed twice withDMF and subsequently three times with methanol. The resin is then vacuumdried briefly while still in the reaction vessel and then transferred toa new, tared vessel for vacuum drying (greater than 12 hours) until itis freely flowing. The mass of crude peptide synthesized is determinedby weighing the vessel containing dried resin, subtracting the mass ofthe tared vessel and adjusting for the resin mass. Expected mass yieldsrange from 60%-90%. Any synthesis that failed to produce at least 200 mgcrude peptide is terminated. The dried resin may be stored at 4° C. forup to 28 days prior to initiation of cleavage.

The cleavage reaction is conducted in a single room. Prior to transferof the set of patient-specific dried resins from the synthesis room tothe cleavage room, the cleavage room is fully qualified by QA forsynthesis of a new GMP product. Qualification includes line clearanceinspection, verification of GMP suite cleaning, staging of all requiredmaterials and glassware, verification of equipment suitability andlabeling, and verification that all required personnel are properlytrained and qualified to conduct the work and are properly gowned andfree of apparent illness.

Room readiness operations initiates with verification of the equipmentto be used (rotary evaporator, vacuum pump, balance) and inspection ofdocumentation indicating that the equipment has been properly cleanedand calibrated (if appropriate). A complete list of all raw materials(TFA, triisopropylsilane (TIS) and 1,2-ethanedithiol) required is issuedby QA and manufacturing identifies lot number to be utilized, retest orexpiration date and quantity of material dispensed for each day'sreactions.

Cleavage of the peptide chain from the resin and cleavage of the sidechain protecting groups are accomplished under acidic conditions (95%TFA) in the presence of 2% triisopropylsilane (TIS) and 1%1,2-ethanedithiol as scavengers of acid-generated free-radicals for 3 to4 hours at room temperature.

Resin is separated from free crude peptide by filtration. The finalsolution of released and de-protected peptide undergoes precipitationwith ether and the precipitate is freeze-dried for 12 hours. The yieldof released crude peptide is determined by weighing the freeze-driedpowder and calculating the ratio of released crude peptide/resin-boundpeptide. Expected yields of crude peptide are 200 mg to 1000 mg. Anycleavage reaction that fails to yield at least 200 mg crude peptide isterminated. The crude peptide is then transferred to the purificationsuite.

The purification is conducted in a single room. Prior to transfer of theset of dried crude peptide from the cleavage room to the purificationroom, the purification room is fully qualified by Quality Assurance forsynthesis of a new GMP product. Qualification includes line clearanceinspection, verification of GMP suite cleaning, staging of all requiredmaterials and glassware, verification of equipment suitability andlabeling, and verification that all required personnel are properlytrained and qualified to conduct the work and are properly gowned andfree of apparent illness.

Room readiness operations initiates with verification of the equipmentto be used (preparative Reverse-Phase High-Performance LiquidChromatography [RP-HPLC], balance, analytical Liquid Chromatography/MassSpectrometer (LC/MS), lyophilizer, balance) and inspection ofdocumentation indicating that the equipment has been properly cleanedand calibrated (if appropriate). A complete list of all raw materials(trifluoroacetic acid [TFA], acetonitrile [ACN], water) required isissued by QA and Manufacturing identifies lot number to be utilized,retest or expiration date and quantity of material dispensed for eachday's reactions.

Purification is initiated by dissolving no more than 200 mg of thefreeze-dried released peptide in ACN. The sample is then further dilutedwith water to 5%-10% ACN. TFA is added to a final concentration of 0.1%.One C-18 RP-HPLC column (10 cm×250 cm) is freshly packed prior to theinitiation of each set of patient specific peptides. Columns areextensively washed with 5% acetonitrile containing 0.1% TFA prior toloading patient peptide. Maximum amount of peptide loaded onto a singlecolumn is 200 mgs. Columns are monitored by UV observance at 220 nm.Following loading of single peptide, the sample is allowed to enter thecolumn and column is washed with 5% acetonitrile/0.1% TFA. A 10%-50%gradient of acetonitrile with 0.1% TFA is used to elute the peptide.Fractions is collected (50 ml each) beginning at the point UV observanceis at 20% above baseline. Fractions continue to be collected until nofurther UV absorbing material is eluting from the column or the gradientis complete. Typically, the main elution peak is separated into 4 to 8fractions.

Each individual fraction is assessed by analytical LC/MS. Analyticalconditions chosen is based on the percent acetonitrile associated withthe peak eluted product. Fractions with the expected mass and puritygreater than or equal to 95% is pooled as peptide product. Typically 2to 4 fractions meet this pooling requirement. The pooled peptide isplaced into a tared jar for freeze-drying and freeze-dried for 24 to 72hours. The mass of lyophilized peptide is determined by determining themass of the jar containing freeze-dried peptide and subtracting the massof the tared jar.

Portions of the freeze-dried peptide is transferred to quality controlfor analysis and disposition. The remaining is stored at −20° C. priorto further processing.

Any peptides for which none of the fractions meet the requirement of 95%purity is discarded. No reprocessing of RP-HPLC fractions can occur. Ifsufficient unpurified freeze-dried and cleaved peptide is available, asecond sample of the peptide may be purified over the column, adjustingthe gradient conditions to improve purity of the eluted peptide.

The column can then be stripped of any remaining peptide by washingextensively with 4 column volumes of 100%0/ACN/0.1% TFA and thenre-equilibrated with 5% ACN/0.1% TFA prior to loading the next peptide.

Peptides for an individual patient is sequentially processed over thesame column. No more than 25 peptides are processed over a singlecolumn.

Unit operations for drug substance manufacturing thus constitute:

Synthesis:

-   -   Condensation, wash and re-condensation for each amino acid    -   Resin washing and vacuum drying    -   Transfer to the cleavage suite

Cleavage:

-   -   Acid cleavage from the resin    -   Separation of released peptide from the resin and peptide        precipitation    -   Transfer to the purification suite

Purification:

-   -   Dissolution in acetonitrile and RP-HPLC purification    -   Freeze-drying of peak fractions for 24 to 72 hours    -   Removal of aliquots for QC testing and storage of remaining        lyophilized product.

Personalized neoantigen peptides may be supplied as a box containing 2ml Nunc Cryo vials with color-coded caps, each vial containingapproximately 1.5 ml of a frozen DMSO/D5W solution containing up to 5peptides at a concentration of 400 ug/ml. There may be 10-15 vials foreach of the four groups of peptides. The vials are to be stored at −80°C. until use. Ongoing stability studies support the storage temperatureand time.

Storage and Stability: The personalized neoantigen peptides are storedfrozen at −80° C. The thawed, sterile filtered, in process intermediatesand the final mixture of personalized neoantigen peptides and poly-ICLCcan be kept at room temperature but should be used within 4 hours.

Compatibility: The personalized neoantigen peptides are mixed with 1/3volume poly-ICLC just prior to use.

Example 8

Formulation Testing

Cloudiness or precipitation was seen with certain peptides in thepeptide pool solution under some conditions. The effect of weak bufferson peptide solubility and stability was therefore evaluated.

It was found that the mixing of poly-ICLC and the peptide pool (in D5Wwith DMSO) sometimes resulted in cloudiness or precipitation, possiblydue to the low pH of the poly-ICLC solution, particularly forhydrophibic peptides. In order to raise the pH of the peptide solution,buffers were tested and the effect on peptide solubility was evaluated.Based on initial testing, citrate and succinate buffers were tested.

It was found that improved solubility was seen for 3 of 4 peptides whichhad solubility issues in D5W alone. Based on this initial observation,19 additional peptides were evaluated with citrate or succinate, and 4further peptides with succinate alone. It was found that solutions of 18of the 19 tested peptides were clear when using either sodium citrate(where tested) or sodium succinate as buffer (none of the four peptidesevaluated in succinate alone demonstrated cloudiness).

Concentrations of 2 mM to 5 mM succinate were found to be effective.Recovery of peptide was improved for one peptide in succinate buffer butnot in citrate buffer. Depending on the peptide pool and theconcentration of succinate buffer used, pH for the peptide solutions inD5W/succinate ranged from about 4.64 to about 6.96.

After evaluation of a total of 27 peptides (including initial difficultto solubilize group of 4 peptides), it was found that one peptidereproducibly showed cloudiness in all conditions, and one additionalpeptide showed slight cloudiness but was fully recoverable uponfiltration. Both of these two peptides had high hydrophobicity.

In general, it was found that peptides that are clear upon dilution to 2mg/ml in D5W with succinate buffer retain clarity upon mixing with otherpeptides (this is generally true for peptides in D5W alone).

In a representative procedure, peptides were weighed and corrected for %peptide content, and then dissolved in DMSO to a concentration of 50mg/mL. The DMSO/Peptide solution was then diluted with 5 mM sodiumsuccinate in D5W to a 2 mg/mL peptide concentration.

Additional peptide solubility conditions were tested. Peptides CS6709,CS6712, CS6720, CS6726, and CS6783 were weighed at approximately 10 mgeach. The peptides were then dissolved in approximately 200 μL USP gradeDMSO to obtain a 50 mg/mL concentration for each peptide. Applicantsobserved that peptide CS6709 at 10.02 mg did not fully dissolve in the200 μL amount of DMSO that was calculated to provide 50 mg/mL. Thesample appeared to be cloudy. Additional 50 μL increments of DMSO wereadded to Peptide CS6709 up to 400 μL; for a total of 600 μL of DMSO.CS6709 went into solution (clear) when the amount of DMSO reached 600μL, the concentration was at 16.67 mg/mL.

To dilute the peptides to 400 μg, a PBS pH 7.4 solution withoutpotassium was prepared. All 5 DMSO peptide samples (50 mg/mL) wereplaced in a single vial for dilution to 400 μg/mL. Each DMSO peptide wasadded to the vial at 40 μL, except for CS6709 which was at aconcentration of 16.67 mg/mL. The volume of CS6709 added to the singlevial was 120 μL. The samples were diluted to 400 μg by adding 4.72 mLPBS pH 7.4. Upon addition of the PBS pH 7.4, it was observed that one ormore of the peptides had precipitated out.

To determine which of the peptides precipitated, Applicants followed thematrix in Table 5 below using very small amounts (10-20 μL) of the DMSOdissolved peptides and adding these peptides to the various liquids.

TABLE 5 Peptide Diluent Matrix PBS 10% D5W (5% Dextrose Peptide LiposomepH 7.4 Water Sucrose USP Grade Inj) CS6709 NP NP NP NP NP CS6712 NP NPNP NP NP CS6720 NP NP NP NP NP CS6726 NP NP NP NP NP CS6783 P P NP NP NPP = precipitation; NP = no precipitation

CS6783 was found to precipitate when PBS pH 7.4 was added as a diluentto the peptide mixture. The Injectable USP grade D5W is a diluentsubstitute for the PBS pH 7.4.

In addition, Applicants tested a small amount of each peptide (<1 mg) tosee if any of the 5 peptides could be dissolved in D5W without usingDMSO. Peptides CS6709, CS6712, CS6720, and CS6726 could be dissolveddirectly in D5W. CS6783 could not be dissolved using D5W.

Example 9

Formulation

Formulations for each patient include up to 20 peptides producedindividually as immunogens. For vaccination, four pools (up to 5peptides each) are prepared for injection into separate sites targetingdistinct parts of the lymphatic system as discussed herein. Theindividual peptides are weighed, dissolved in DMSO at highconcentration, diluted with 5% dextrose in water (D5W) and sodiumsuccinate (4.8-5 mM) and mixed in four pools. The individual pools arefiltered through a 0.2 μm filter to reduce bioburden, aliquoted intovials and frozen. The frozen vials are stored frozen until use.

As described herein, the set of patient-specific peptides constitutingthe drug substances are individually prepared, lyophilized, tested andreleased, and stored following manufacture. To prepare these peptidesfor injection, four groups comprised of up to 5 different peptides eachare identified for pooling.

Example 10

Preparation of Vaccines

Weighing and Dissolution:

Based on gross weight and peptide content, 15 mg (net weight) orslightly more of each individual peptide are weighed and 100% USP GradeDMSO (2:250 μl) is added to achieve a final peptide concentration of 50mg/ml. Based on developmental studies, >95% of the dissolved peptidesdemonstrate clarity at this point.

Dilution and Mixing:

USP Grade D5W containing 5 mM Sodium Succinate (D5W/Succ) is preparedand filtered (0.2 J.tm) for use as diluent. 250 μl each dissolvedpeptide is diluted with D5W/Succ to reduce the peptide concentration to2 mg peptide/ml and adjust pH to approximately ˜6.0. Any peptides thatdo not demonstrate a clear solution are replaced with another peptide(or D5W/Succinate solution only if no additional peptides areavailable). 5.5 ml of each diluted peptide solution is then combinedinto a single 5-peptide containing pool with each peptide at aconcentration of 400 μg peptide/ml. The first of two 0.2 μm membranefiltration steps are then performed. Each pool is drawn into a 60 mlBecton Dickson (or equivalent) syringe fitted with a leur lock tip andan 18 gauge blunt needle. The needle is removed and replaced with a 25mm PALL PES (Polyether sulfone) 0.2 μm membrane filter (PALL CatalogHP1002). The contents of the syringe are transferred through the filterinto a 50 ml sterile polypropylene tube (Falcon#352070 or equivalent).An aliquot of each pool is removed for testing and the remainder frozenat −80° C. The remainder of each individual diluted peptide is stored at−20° C. until all analysis is complete.

Shipping:

The frozen peptide pools are shipped using validated shipping containersand overnight air.

Filtration and Storage:

The frozen pools are thawed and transferred to a biosafety cabinet. A 2ml sample from the thawed pool is tested for sterility and endotoxintesting. The remaining bulk solution are processed for a second of two0.2 μm membrane filtration steps. The bulk pooled peptide is drawn intoa Becton Dickinson (or equivalent) 60 ml syringe fitted with a luer-locktip and an 18 gauge blunt needle. The needle is removed and replacedwith a 25 mm PALL PES (Polyether sulfone) 0.2 μm membrane filter (PALLCatalog HP1002). The contents of the syringe are transferred through thefilter into a 50 ml sterile polypropylene tube (Falcon#352070 orequivalent). 1.5 ml aliquots of the peptide solution are thentransferred aseptically into fifteen pre-labeled sterile 1.8 ml NuncCryo vials (Cat #375418). The vials are capped with one of 4 color-codedcaps. A different color-coded cap is used for each of the 4 pools ofpeptides for a single patient to assist identification. The vials arelabeled with the patient's name, medical record number study number,original product/sample alphanumeric identifier and the uniquealphanumeric identifier (A-D). All vials are frozen at −80° C. Theremaining frozen vials are stored until all release testing has passedacceptance criteria. Patients are not scheduled for immunization untilall release testing is complete and product is released to the pharmacy.

Alternatively, on each day of immunization, one set (four) of vialswhich have not yet been subjected to sterilizing filtration within abiosafety cabinet as described herein are thawed and transferred to abiosafety cabinet. The contents of each vial are withdrawn into separatesyringes. A 0.2 μm sterilizing filter is attached and the contentstransferred through the filter into a sterile vial. The filter isremoved and checked for integrity. 0.75 ml of the peptide mixture isthen withdrawn using a sterile syringe and mixed by syringe-to-syringetransfer with 0.25 ml poly-ICLC (Hiltonol®).

Analysis:

Three tests (Appearance, Identity and Residual Solvents) are conductedas in-process tests on an aliquot of the pooled peptides. Endotoxin istested on an aliquot of the thawed peptide pool prior to finalfiltration. Sterility is analyzed on the combined samples from two vialsof the final product. This approach is taken to assure that the keybiochemical information (peptide solubility, identity of each peak ineach pool and levels of any residual solvents) is available prior toconducting the final filtration. Upon receipt of pooled and filteredbulk peptide pools, endotoxin testing and culturing for microorganismsis performed to evaluate microbiological purity. Meeting the endotoxinspecification is required for product use. Any positive results in themicrobial culture test is investigated for impact on product use. Thekey safety test, sterility, is conducted on vialed samples after finalfiltration and vialing, the samples closest to patient use.

Example 11

Administration

Following mixing with the personalized neo-antigenicpeptides/polypeptides, the vaccine (e.g., peptides+poly-ICLC) is to beadministered subcutaneously.

Preparation of personalized neo-antigenic peptides/polypeptides pools:peptides are mixed together in 4 pools of up to 5 peptides each. Theselection criteria for each pool is based on the particular MHC alleleto which the peptide is predicted to bind.

Pool Composition:

The composition of the pools will be selected on the basis of theparticular HLA allele to which each peptide is predicted to bind. Thefour pools are injected into anatomic sites that drain to separate lymphnode basins. This approach was chosen in order to potentially reduceantigenic competition between peptides binding to the same HLA allele asmuch as possible and involve a wide subset of the patient's immunesystem in developing an immune response. For each patient, peptidespredicted to bind up to four different HLA A and B alleles areidentified. Some neoORF derived peptides are not associated with anyparticular HLA allele. The approach to distributing peptides todifferent pools is to spread each set of peptides associated with aparticular HLA allele over as many of the four pools as possible. It ishighly likely there are situations where there are more than 4 predictedpeptides for a given allele, and in these cases it is necessary toallocate more than one peptide associated with a particular allele tothe same pool. Those neoORF peptides not associated with any particularallele are randomly assigned to the remaining slots. An example is shownbelow:

A1 HLAA0101 3 peptides A2 HLA A1101 5 peptides B1 HLA B0702 2 peptidesB2 HLA B6801 7 peptides X NONE (neoORF) 3 peptides Pool # 1 2 3 4 B2 B2B2 B2 B2 B2 B2 A2 A2 A2 A2 A2 A1 A1 A1 B1 B1 X X X

Peptides predicted to bind to the same MHC allele are placed intoseparate pools whenever possible. Some of the neoORF peptides may not bepredicted to bind to any MHC allele of the patient. These peptides arestill utilized however, primarily because they are completely novel andtherefore not subject to the immune-dampening effects of centraltolerance and therefore have a high probability of being immunogenic.NeoORF peptides also carry a dramatically reduced potential forautoimmunity as there is no equivalent molecule in any normal cell. Inaddition, there can be false negatives arising from the predictionalgorithm and it is possible that the peptide contains a HLA class IIepitope (HLA class II epitopes are not reliably predicted based oncurrent algorithms). All peptides not identified with a particular HLAallele are randomly assigned to the individual pools. The amounts ofeach peptide are predicated on a final dose of 300 μg of each peptideper injection.

For each patient, four distinct pools (labeled “A”, “B”, “C” and “D”) of5 synthetic peptides each are prepared by the manufacturer and stored at−80° C. On the day of immunization, the complete vaccine consisting ofthe peptide component(s) and poly-ICLC is prepared in the researchpharmacy. One vial each (A, B, C and D) is thawed at room temperatureand moved into a biosafety cabinet for the remaining steps. 0.75 ml ofeach peptide pool is withdrawn from the vial into separate syringes.Separately, four 0.25 ml (0.5 mg) aliquots of poly-ICLC is withdrawninto separate syringes. The contents of each peptide pool containingsyringe is then gently mixed with a 0.25 ml aliquot of poly-ICLC bysyringe-to-syringe transfer. The entire one ml of the mixture is usedfor injection. These 4 preparations are labeled “study drug A”, “studydrug B”, “study drug C”, and “study drug D”.

On each day of immunization, patients are subcutaneously injected withup to four pools of personalized neoantigen peptides mixed withpoly-ICLC (Hiltonol®).

-   -   The injection volume for each mixture of peptides and Hiltonol®        is 1 ml.    -   Each pool of peptides consists of up to 5 peptides, each at a        concentration of 400 μg/ml.    -   The composition of the peptide pool is;        -   Up to five peptides each at a concentration of 400 μg/ml.        -   4% DMSO        -   4.8-5% dextrose in water        -   4.8-5 mM Sodium Succinate    -   Hiltonol® consists of:        -   2 mg/ml poly I:poly C        -   1.5 mg/ml poly-L-Lysine        -   5 mg/ml sodium carboxymethylcellulose        -   0.9% sodium chloride

Each 1 ml injection volume consists of 0.75 ml of one of the fourpeptide pools mixed with 0.25 ml Hiltonol®. Following mixing, thecomposition is:

-   -   Up to five peptides each at a concentration of 300 μg/ml.    -   ≤3% DMSO    -   3.6-3.7% dextrose in water    -   3.6-3.7 mM Sodium Succinate    -   0.5 mg/ml poly I:poly C    -   0.375 mg/ml poly-L-Lysine    -   1.25 mg/ml sodium carboxymethylcellulose    -   0.225% sodium chloride

Injections:

At each immunization, each of the 4 study drugs is injectedsubcutaneously into one extremity. Each individual study drug isadministered to the same extremity at each immunization for the entireduration of the treatment (i.e. study drug A will be injected into leftarm on day 1, 4, 8 etc., study drug B will be injected into right arm ondays 1, 4, 8 etc.). Alternative anatomical locations for patients whoare status post complete axillary or inguinal lymph node dissection arethe left and right midriff, respectively.

Vaccine is administered following a prime/boost schedule. Priming dosesof vaccine is administered on days 1, 4, 8, 15, and 22 as shown herein.In the boost phase, vaccine is administered on days 85 (week 13) and 169(week 25).

All patients receiving at least one dose of vaccine is evaluated fortoxicity. Patients are evaluated for immunologic activity if they havereceived all vaccinations during the induction phase and the firstvaccination (boost) during the maintenance phase.

Example 12

Short-Term Room Temperature Stability of Final Dosage Form

Peptide Stability.

A peptide pool (Pool 3) consisting of the five peptides shown in Table 6below was prepared by dissolution in DMSO and dilution withD5W/Succinate (2 mM) to 2 mg/ml and pooling to a final peptideconcentration of 400 μg per ml and a final DMSO concentration of 4%.After preparation, peptides were filtered with a 25 mm Pall PES filter(Cat#4612) and dispensed into Nunc Cryo vials (#375418) in one mlaliquots.

TABLE 6  Peptides and sequences of Pool 3 Frac Total Hydro- ydro-Peptide Sequence % Peptide Content AA phobic Hphobic 1 CS6919KLAWRGRISSSGCPSMT 30 9 0.30 SPPSPMFGMTLHT 2 CS6931 VAGLAASGLHGSAWLVP 3011 0.37 GEQPVSGPHHGKQ 3 CS6934 SKRGVGAKTLLLPDPFL 29 11 0.38 FWPCLEGTRRSL4 CS6941 AHRQGEKQHLLPVFSRL 29 12 0.41 ALRLPWRHSVQL 5 CS7416AESAQRQGPNGGGEQSA 20 4 0.20 NEF

Three samples were prepared by mixing 0.75 ml of Pool3 with 0.25 mlHiltonol® as planned for the dosage form preparation. The samples werethen left at room temperature for 0, 4 and 6 hours and analyzed byRP-HPLC (Table 7). No change was noted for 4 of the 5 peptides. Asslight increase in a second peak associated with peptide CS6919 wasnoted, increasing from 14% to 17% and 18% at 4 and 6 hours,respectively. As noted in a −20° C. stability study, peptides CS6919 andCS6934 (both represented in Pool4) can form a heterodimer (as shown bymass spectrometry) which elutes at the position of this impurity.Recovery of all peptides was above 90%, indicating no breakdown and lossof any peptides in the final dosage form after 6 hour room temperatureincubation.

TABLE 7 Summarized Stability of Pool 3 after Mixing with Hiltonol ® andRoom Temperature Incubation T0 Pool 3 + Main Total Total Hiltonol PeakImpurities Peak % Purity % Impurity CS6919 7786.28 1256.72 9043 86.113.9 CS6931 9014.82 198.6 9213.42 97.84 2.16 CS6934 6147.14 244.496391.63 96.17 3.83 CS7416 5988.42 143.98 6132.4 97.65 2.35 CS69417140.91 0 7140.91 100 0 Pool 3 + Main Total Total Recovery Hiltonol PeakImpurities Peak % Purity % Impurity Main Peak Total AUP 4 Hours RTCS6919 7238.56 1492.4 8730.96 82.91 17.09  93%  97% CS6931 8523.53265.54 8789.07 96.98 3.02  95%  95% CS6934 5842.22 184.46 6026.68 96.943.06  95%  94% CS7416 5669.85 148.82 5818.67 97.44 2.56  95%  95% CS69416676.54 0 6676.54 100 0  93%  93% 6 Hours RT CS6919 7688.89 1703.99392.79 81.86 18.14 106% 108% CS6931 9387.81 311.37 9699.18 96.79 3.21110% 110% CS6934 6268.16 221.46 6489.62 96.59 3.41 107% 108% CS74166197.48 132.83 6330.31 97.9 2.1 109% 109% CS6941 7158.29 0 7158.29 100 0107% 107%

Poly-ICLC Stability.

In a second study, another peptide pool (Pool4) was used, mixed withHiltonol® (0.75 ml peptide pool+0.25 ml Hiltonol®) and stored at roomtemperature for 6 hours. The room temperature incubatedpeptide+Hiltonol® mix and Hiltonol® alone (that was stored continuouslyat 4° C.), were then diluted to 20 ug/ml poly-ICLC and assayed for TLRstimulation using mouse dendritic cells according to published methods.After 24 hour stimulation, quantitative PCR was used to assess thelevels of induction of a number of key immune markers as shown in FIG.6. There was no difference in the stimulatory capability of poly-ICLCafter 6 hour room temperature with peptide pools in the finalformulation, indicating that Hiltonol® was not affected by anyformulation components (DMSO[4%], D5W, 5 mM Succinate, peptides) and wasstable in the final dosage form for up to 6 hours at room temperature.

Example 13

Lyophilization of the Final Formulation Form

The formulation for peptides is as following: Each pool of peptidesconsists of up to 5 peptides, each at a concentration of 400 μg/ml. Thecomposition of the peptide pool is:

Up to five peptides each at a concentration of 400 μg/ml

4-8% DMSO

4.6-4.8% dextrose in water

5 mM Sodium Succinate

The bulking agent that is used for stabilization is Dextrose in water(D5W). The final formulation is based on the thermal properties of theformulation matrix. Modulated differential scanning calorimetry (MDSC)data suggested the presence of two glass transition temperatures (Tg′)at −24° C. and −56° C. respectively and an exothermic reaction at −67°C. due to melting of DMSO. Based on the literature, the glass transitionof D5W is −43° C. The MDSC data suggests that the presence of DMSOfurther reduces the glass transition temperature. Based on thisinformation, the lyophilization feasibility of peptides was checkedusing two additional bulking agents, Sucrose and Trehalose. Thefollowing formulations were assessed with MDSC analysis (FIG. 7-9):

1. 5% D5W and 0.8% DMSO

2. 10% Sucrose and 0.8% DMSO

3. 10% Trehalose and 0.8% DMSO

The above formulations were lyophilized using a conservative lyo cycleby freezing −50° C. for 3 hrs, primary drying at −35° C. at 75 mtorr for30 hrs and at −30° C. for 30 hrs (FIGS. 10 and 11). The formulationcontaining D5W-DMSO collapsed completely, though partial cake is seenfor the formulation containing D5W alone. The lyophilization resultssuggest that in presence of 0.8% DMSO, the formulation containingtrehalose or sucrose is more compatible for lyophilization thanformulation containing dextrose (FIG. 12).

The samples (25 μL) were analyzed by MDSC using the following program.The following parameters were used to monitor thermal events:

1. Equilibrate at 20.00° C.

2. Isothermal for 5.00 min

3. Modulate+/−1.00° C. every 60 seconds

4. Data storage: ON

5. Ramp 1.00° C./min to −70° C.

6. Equilibrate at −70° C.

7. Isothermal for 5.00 min

8. Ramp 1.00° C./min to 20.00° C.

9. Equilibrate at 20.00° C.

10. Data storage: OFF

11. Isothermal for 5 minutes

12. End of Method

Lyophilization.

MDSC was used to determine the glass transition temperature (T_(g))which is used to select the primary drying and freezing temperature ofthe products (Table 8 and FIG. 7-9). The data indicates that melting ofDMSO occurs around −68° C. in all formulations. There were two glasstransitions for all 3 formulations. The formulation containing dextrose,trehalose or sucrose has the lowest heat flow glass transition of −59°C., −42° C. and −50° C. respectively suggesting that it is difficult tolyophilize the formulation containing D5W-DMSO without collapse/melt.

TABLE 8 MDSC analysis of 10% Sucrose and 0.8% DMSO Freezing DMSO tempmelting melting Tg'1 Tg'2 Formulations (° C.) (° C.) (° C.) (° C.) (°C.) 5% D5W-0.8% DMSO −18.2  −0.38 −67.86 −24.27 −59.17 heat flow 5%D5W-0.8% DMSO N/A N/A NA −33.31 −62.86 reverse heat flow 10%Trehalose-0.8% −12.64 −1.25 −68.06 −24.4  −42.55 DMSO heat flow 10%Trehalose-0.8% N/A N/A NA −24.4  −39.2  DMSO reverse heat flow 10%Sucrose-0.8% −11.36 −0.26 −67.87 −23.53 −50.31 DMSO heat flow 10%Sucrose-0.8% N/A N/A NA −31.21 N/A DMSO reverse heat flow

Lyophilization was initially tried with Nunc vials, and it was foundthat the configuration of the nunc vials was not adequate to lyophilizethe formulation matrix. One ml of a 5% D5W and 0.8% DMSO formulation infour 1.8 mL sterile Nunc vials (Thermo Scientific) was lyophilized usingthe lyophilization cycle (Freezing to −50° C. and hold for 2 hrs,primary drying at −15° C. for 20 hrs at 75 m torr and 8 hrs of secondarydrying at 20° C. with 75 m torr pressure). It was observed that therewas no cake in the vials and the liquid residual DMSO and D5W in theform of small liquid droplets were noticed at the bottom of the Nuncvials.

The flint vial suitable for lyophilization was chosen to determine thefeasibility of lyophilization of the lead formulation. Five vialscontaining 1.5 ml of each formulation were filled in 3 mL 13 mm flintvials and partially closed with a 13 mm lyo stopper and kept in themiddle shelf of Lyostar II for lyophilization

It is difficult to lyophilize a formulation having a glass transitionbelow −50° C. Based on the glass transition temperature, the followingconservative lyophilization parameters were set for lyophilization(Table 9). The results obtained on pressure profile and temperatureprofiles are presented in FIGS. 10 and 11 respectively. The piranipressure reached below shelf set pressure during primary and secondarydrying suggesting there is no moisture in the chamber (FIG. 10) and thelyophilization cycle is complete.

TABLE 9 Lyophilization parameters of placebo formulation of peptidescontaining DMSO and Tehalose, Sucrose or D5W. Ramp/ Hold Hold Time RampTime Step Temperature Pressure (minutes) Rate (min or hr) Load   20° C.atmosphere N/A N/A Freezing −50° C. N/A N/A 1° C./ 70 (Ramp) minFreezing −50° C. N/A 120 N/A 180 (Hold) Primary −35° C. 75 m torr 18001° C./ 15 (Ramp) drying min Primary −30° C. 75 m torr Till pirani 1° C./5 ramp drying reaches min 75 m torr (1800 min) Secondary   20° C. 75 mtorr N/A 1° C./ 50 (ramp) drying min Secondary   20° C. 75 m torr Tillpirani NA Till pirani drying reaches reaches 75 m torr 75 m torr (1800(220 min) min) Backfill to 600 Torr under Nitrogen, and stopper, Bringto 760 (Atmos), crimp and seal,

Physical Appearance of the Cake.

The formulation containing D5W and DMSO is completely collapsed andmelted, whereas the formulation containing Trehalose-DMSO orSucrose-DMSO has white amorphous cake with slight collapse (FIG. 12).

Example 14

Algorithm for Producing Soluble Peptides in D5W/Succinate or OtherAqueous Buffers

Applicants developed an algorithm for accurate prediction of solubilityof peptides in various aqueous solutions. It is generally recognizedthat solubility of any given peptide in aqueous solutions is difficultto predict based on sequence information alone and often requiresempirical determination. Using two calculable parameters that relate tohydrophobicity and the isoelectric point, Applicants have identifiedthat peptides with particular calculable combinations of theseparameters exhibit high or low solubility, thus providing a solution tothe problem of predicting peptide solubility.

The isoelectric Point (Pi) can be estimated using calculators readilyavailable on the internet (for example, seewww.geneinfinity.org/sms/sms_proteiniep.html) or can be easilycalculated using the known pH/charge formulas for all potential chargedamino acids. The pKa's of the side chains of the charged amino acids (H,R, K, D, E, C, Y) and of the peptide amino and carboxy terminus areknown (Table 10).

TABLE 10 (NH2—) 9.69 (—COOH) 2.34 K (Lysine) 10.5 D (Aspartic acid) 3.86R (Arginine) 12.4 E (Glutamic acid) 4.25 H (Histidine) 6.00 C (Cysteine)8.33 Y (Tyrosine) 10.0

Lehninger, Biochemistry

The actual charge of each amino acid will depend on the pH of thesolution according to the formulas:

For  NH 2, K, R, H${Z({charge})} = \frac{10^{pKa}}{\left( {10^{pH} + 10^{pKa}} \right)}$For  —COOH, D, E, C, Y${Z({charge})} = \frac{10^{pH}}{\left( {10^{pH} + 10^{pKa}} \right)}$

The net charge on the peptide at any given pH is the sum of the chargeson each individual amino acid or termini. The isoelectric point is thepH at which the net charge is 0.

Hydrophobicity can be calculated in various ways. One way to calculatehydrophobicity is to look for regions of each peptide that arehydrophobic and to calculate an index for the degree of hydrophobicityof each region and find the region with the highest degree ofhydrophobicity. This parameter can be designated HYDRO. This calculationcan be readily accomplished by using published values of hydrophobicity(or hydrophilicity) for each amino acid side chain, identifyinguninterrupted stretches of hydrophobic amino acids in the peptide andsumming the hydrophobicity of each amino acid in each region. As anexample, the following table of hydrophilicites for each amino acid aregiven (Table 11):

TABLE 11 Alanine −0.5 Cysteine −1 Aspartic Acid 3 Glutamic acid 3Phenylalanine −2.5 Glycine 0 Histidine −0.5 Isoleucine −1.8 Lysine 3Leucine −1.8 Methionine −1.3 Asparaginine 0.2 Proline 0 Glutamine 0.2Arginine 3 Serine 0.3 Threonine −0.4 Valine −1.5 Tryptophan −3.4Tyrosine −2.3Hydrophobic amino acids have negative values.

Each amino acid is assigned it's hydrophilicity value and for eachcontiguous stretch of amino acids which all have values less than 0,these values are summed together and this sum is the hydrophobicityindex for the given contiguous stretch. The most hydrophobic stretch isthe one with the most negative value. This value defines the parameterHYDRO. An example of these values for an example peptide is shown (FIG.13). The values in blue represent the hydrophilicity value (negativevalues thus represent hydrophobic residues) for each amino acid andvalues in red indicate the sum of hydrophobic values across thehydrophobic stretch.

When these two parameters (P_(i) and HYDRO) are examined together,peptides with certain combined characteristics are more commonly solublewhile with other combined characteristics are insoluble. These combinedcharacteristics can thus be used during the process of designing apeptide for synthesis so that the likelihood of the peptide beingsoluble in the formulation buffer after synthesis is increased.

Table 12 displays the calculated P_(i) and HYDRO values for 221 peptidesand whether the peptide is soluble or insoluble in the 5% Dextrose inWater (D5W)/5 mM succinate formulation as described herein.

TABLE 12  Soluble/ Peptide Sequence Pi Insoluble HYDROTSGSSTALPGSNPSTMDS 2.925 I −2.7 GSGD DGVSEEFWLVDLLPSTHY 3.585 I −9.2 TDVTYDGHPVLGSPYTVEA 3.695 I −4.2 SL EYWKVLDGELEVAPEYPQ 3.815 I −5.7STARDWL GLEQLESIINFEKLTEWTSS 3.795 I −3.8 SERYIGTEGGGMDQSILFL 4.005 I−8.4 AEEGTAK TTTSVKKEELVLSEEDFQG 4.005 I −5.1 ITPGAQ EEFNRRVRENPWDTQL4.125 I −14 WMAFVAFQDE EDSKYQNLLPFFVGHNM 4.155 I −6.5 LLVSEETTSGDERLYPSPTFYIHEN 4.155 I −7.5 YLQLFE ESKLFGDPDEFSLAHLLEP 4.275 I −6.4FRQYYL TISLLLIFYNTKEIARTEEH 4.705 I −12 QE ETYSRSFYPEHSIKEWLIG 4.705 I−7.3 MELVFV TLDDIKEWLEDEGQVLNI 4.755 I −5.2 QMRRTLHK NHSAKFLKELTLAMDELE4.765 I −5.8 ENFRG KAHVEGDGVVEEIIRYHPF 4.785 I −6.6 LYDRETEAAFSVGATGIITDYPTAL 5.115 I −4.6 RHYLDNHG IGALNPKRAAFFAEHYES 5.395 I−6.5 WE ERLSIQNFSKLLNDNIFYM 6.935 I −7.9 S LDVLQRPLSPGNSEFLTAT 6.935 I−6.1 ANYSK SAVSAASIPAMHINQATN 7.845 I −4.1 GGGS ISSLFVSYFLYRVVFHFE 7.695I −8.9 LVDQWRWGVFSGHTPP 7.695 I −9.1 SRYNFDWWY DHAPEFPAREMLLKYQKL 7.155I −6.6 LCQERYFL SVLREDLGQLEYKYQYAY 7.595 I −7.6 FRMGIKHPDADRRRQRSTFRAVLHFVE 7.855 I −8.3 GGESEE AIYHKYYHYLYSYYLPASLK 9.075 I−11.5 NMVD KQGWTTEGIWKDVYIIKL 9.555 I −7.4 AIISSLFVSYFLYR 9.585 I −8.9SGQPAPEETVLFLGLLHGL 10.795 I −9 LLILRRLRGG KQYLDHSGNLMSMHNIK 10.175 I−8.1 IFMFQLLRG SMWKGELYRQNRFASSK 10.195 I −4.7 ESAKLYGSLRVFIGNIAVNHAPVSLRP 12.405 I −5.8 GLGLPPGAPPGTVP DVGVNSLQQYYLSPDLHF3.965 I −6.4 SLIQKENLD DHVSIILLSATIPNALEFAD 3.695 I −7.2 WIGDPDVGVNSLQQYYLSPDL 3.595 I −6.4 HFSLI LHFIMPEKFSFWEDFEE 3.995 I −7.9DPLMTCSEPERLTEILFQR 3.885 I −6.1 AELE TLKEEVNELQYRQKQLELL 4.795 I −5.8ITNLMRQVD LKEMNEKVSFIKNSLLSLD 5.385 I −4.3 SQVGHLQD YFDVVERSTEKIVDTSLIFN4.065 I −6.1 I VARNYLREAVSHNASLEV 7.765 I −5.6 AILRD AAAFPSQRTSWEFLQSLV9.885 I −4.3 SIKQEKPA NNGPVTILQRIHHMAAS 11.045 I −5.5 HVNITSLMSNLAFADFCMRMYL 6.085 I −5.4 YRMYQKGQETSTNLIASIF 9.525 I −4.8 APAAGDFIRFRFFQLLRLER 11.925 I −5 FF LNYLRTAKFLEMYGVDLH 7.635 I −4.3 PVYGFKMDRQGVTQVLSCLSYI 8.875 I −4.1 SALGMMT LTKLKFSLKKSFNFFDEYF 9.955 I −5LLTDRNTSGTTFTLLGVSDYP 3.705 S −12.4 ELQVPLFLVFLA DSAVDKGHPNRSALSLTPGL10.085 S −12.4 RIGPSGLFLVFLA ALSLTPGLRIGPSGLFLVFLAE 10.085 S −12.4SAVDKGHPNRS PIDTSKTDPTVLLFMESQYS 3.505 S −9.3 QLGQD NNSKKKWFLFQDSKKIQVE10.385 S −10.2 QPQ SKRGVGAKTLLLPDPFLFWP 10.565 S −10.2 CLEGTRRSLSLPKSFKRKIFVVSATKGVPA 10.985 S −7.3 GNSD DNHLRRNRLIVVDLFHGQL 10.795 S−6.6 TKRQVILLHTELERFLEYLPLR 9.715 S −7.8 F TKDRDLLVVAHDLIWKMSP 9.755 S−7.6 RTGDAKPS HRPRPFSPGKQVSSAPLFML 10.385 S −7.4 DLYNPENDDLFMMPRIVDVTSLA 3.425 S −6.9 TEGG RPAGRTQLLWTPAAPTAM 10.885 S −7.4AEVGPGHTP DPNKYPVPENWLYKEAHQL 4.625 S −7.5 FLE SHTQTTLFHTFYELLIQKNKH10.045 S −10.8 K DGGRQHSGPRRHSGAGPK 10.095 S −10.3 PSSSEWAVCWAPSTLPVISDSTTKRRWSALVIG 11.325 S −5.6 L GSYLVALGAHTGEES 4.245 S −7.9RARQILIASHLPFYELRHNQV 9.835 S −5.9 ES LPVFIGNIAVNHAPVSLRPG 11.045 S −5.8LGLPPGAPPGTVP VAGLAASGLHGSAWLVPGE 8.055 S −7.2 QPVSGPHHGKQDASDFLPDTQLFPHFTELLLP 3.315 S −5.4 LDPLEGSSV DRSVLAKKLKFVTLVFRHGD 10.805S −10.2 RSPID VEQGHVRVGPDVVTHPAFL 6.025 S −6.3 V SQSSTPAMLFPAPAAHRTLT9.845 S −6.7 YLSQ GTKALQLHSIAGRWPRMEP 10.085 S −6.4 WVVESMSLGVPTIKNSDKNVVLEHFG 7.795 S −4.8 RLVLGKFGDLTNNFSSPHAR 11.325 S −5.1YLLPKTAVVLRCPALRVRKP 11.405 S −5.9 LENNANHDETSFLLPRKESN 4.275 S −6.1 IVDKKNITNLSRLVVRPDTDAVY 10.175 S −4.8 GQSFFVRNKKVRTAPLSEGP 11.465 5 −6.5HSLG KMQRRNDDKSILMHGLVSL 11.305 S −5.4 RESSRG HKSIGQPKLSTHPFLCPKPQ10.555 S −5.3 KMNTSLGQHLTL NTDKGNNPKGYLPSHYKRV 10.195 S −4.9 QMLLSDRFLWDGPPENDMLLKEICGSLIP  3.585 S −4.9 PRVDLQGAELWKRLHEIGTE 7.795 S −5.3MIITK DHAPEFPAREMLLKYQKLLS 7.725 S −4.9 QER SSELTAVNFPSFHVTSLKLM 7.815 S−4.9 VSPTS EVVGGYTWPSGNIYQGYW 9.395 S −6.2 AQGKR GSTLSPVPWLPSEEFTLWSS3.125 S −8.1 LSPPG GSGALGAVGATKVPRNQD 10.085 S −5.2 WLGDQYKATDFVADWAGTFK 4.345 S −5.7 MVFTPKDGSG LSPREEFLRLCKKIMMRSIQ 10.565 S−4.4 GALGAVGATKVPRNQDWL 12.135 S −5.2 GVSRQLRTKA VQLSIQDVIRRARLSTVPTA12.575 S −5.2 QRVALRSGWI AVGATKVPRNQDWLGVSR 11.325 S −5.2 QLGAVGATKVPRNQDWL 10.085 S −5.2 EGPMHQWVSYQGRIPYPR 9.555 S −4.9 PGMCPSKTAHRQGEKQHLLPVFSRLALR 12.405 S −4.1 LPWRHSVQL KLAWRGRISSSGCPSMTSPP 11.325S −5.7 SPMFGMTLHT SLTEESGGAVAFFPGNLSTSS 3.125 S −7.5 SAAQRKLYQDVMHENFTNLLS 7.885 S −4.1 VGHQP DDSLHIQATYISGPVLAGSG 3.595 S −5 DSRNTGHLHPTPRFPLLRWT 10.795 S −3.8 QEPQPLE SHNELADSGIPENSFNVSSL 3.685 S−3.3 VE VPRIAELMNKKLPSFGPYLE 9.625 S −4.1 KHLPGVNFPGNQWNPVEG 7.815 S−3.6 ILPS GRMSPSQFARVPGYVGSPL 11.385 S −4.1 AAMNPKLPDEVSGLEQLESIINFEKLTE 3.435 S −3.8 WTSSNVME DATFSDGSLGQLVKNTSATY 3.885S −5.5 ALS DEQGREAELARSGPSAAGP 7.205 S −3.3 VRLKPGLVPGL RRGGALFASRPRFTPL12.875 S −5.3 SAAEALELNLDEESIIKPVHSS 3.885 S −3.6 ILGQEPGGDSGELITDAHELGVAHP 4.055 S −4 PGY PETGEIQVKTFLDREQRESYE 4.495 S −4.7LKV VSGLEQLESIINFEKL 3.965 S −3.6 GLEQLESIINFEKL 3.965 S −3.6LPDEVSGLEQLESIINFEKL 3.585 S −3.6 TTVTHERKQAKVVNPPIQEV 10.965 S −3.2GKGARK RYNSTAATNEVSEVTVFSKS 7.015 S −5.9 PVT KGEKNGMTFSSTKDYVNNV 9.555 S−4.2 VSWGKKVQPIDSILADWNE 3.825 S −4.1 DIEAFEMMEKD GHQKLPGKIHLFEAEFTQVA7.895 S −6.6 KKEPDG TSRRLTGLLDHEVQAGRQ 10.795 S −3.6SPIKLVQKVASKIPFPDRITEE 9.755 S −3.3 SV RGQIKLADFRLARLYSSEESR 10.375 S−4.1 PLMQTELHQLVPEADPEEM 3.585 S −3.3 A TFPKKIQMLARDFLDEY 6.975 S −4.3LLDILDTAGREEYSAMRDQY 4.205 S −3.6 MRT NILHQEELIAQKKWEIEAKM 5.525 S −4.1EQK VPDINMEKKLRKIRAQTQK 10.285 S −4.6 HLDLYARDG HPEFANPDSMEYISDVVDE3.375 S −4.1 VIQN SEIDFPMARSKLLKKKLPSKD 10.385 S −3.6 LEDSDKLFESKAELADHQKF 4.365 S −4.3 MPPPGALMGLALKKKSIPQ 10.845 S −4.1 PTNSGARIGAPPPHATATSSSSF 7.845 S −3.8 MPGTWGREDL LGETMGQVTEKLQPTYMEE 3.795 S−4 T TWAGHVSTALARPLGAPW 7.155 S −4.3 AEPGSCGPGTN WTPAAPTAMAEVGPGHTP6.015 S −3.8 AHPSQGAVPP EQGPWQSEGQTWRAAGG 6.435 S −3.8 RVPVPCPAAGPGLARDIPPAVTGKWKLSDLRR 10.685 S −3.4 YGAVPSG KGASLDAGWGSPRWTTTR 12.405 S−4.6 MTSASAGRSTRA LSVPFTCGVNFGDSIEDLEI 2.835 S −3.9 VTSPKASPVTFPAAAFPTAS9.885 S −4.4 PANKD DSPAGPRRKECTMALAPNF 10.095 S −5.5 TANNRPSTANYNSFSSAPMPQIPVA 5.925 S −2.5 SVTPT SAVSAASIPAEHINQATNGG 5.125 S−2.3 GS NNQTNSPTTPNFGSSGSFN 3.095 S −2.5 LPNSGD GTEPEPAFQDDAVNAPLEF3.505 S −3 KMAAGSSG TNGPEKNSSSFPSSVDYAAS 9.625 S −3.3 GPRKLPAPPPAVPKEHPAPPAPPPA 7.815 S −2 SAPTP MSQDIKKADEQIESMTYSTE 4.725 S −4RKT PAHPSQGAVPPSRAAAEPH 7.965 S −2.3 LKPSPSELQTA SGSPPLRVSVGDFSQEFSPI3.585 S −2.5 QEAQQD RQRRGRLGLPGEAGLEGFEP 4.725 S −2.5 SDALGPDAESAQRQGPNGGGEQSAN 3.965 S −2.5 EF AAVRPEQRPAARGSRV 12.405 S −2.5FYSNSTVSETQWKVTVTPR 9.715 S −4.8 LMGRLQHTFKQKMTGVGA 11.565 S −3.4 SLEKRVDKNGRRRLVYLVENPGG 10.385 S −8.9 VDKNGRRRLVYLVENPGGY 9.835 S −8.9 VAYSFLLQVPGSPVVSPSA 6.015 S −6.1 FVGKLQRHPVAVDVLL 10.085 S −5.1YPEPQNKEAFVHSQMYSTD 4.055 S −5 YDQI DDNGNILDPDKTSTIALFKA 4.115 S −7 HEVLVGQLKRVPRTGRVYRNVQ 12.235 S −3.8 RPESVS PASRALEEKKGNYVVTDHG 7.155 5−5.7 SCV LCPASRALEEKKGNYVVTDH 7.155 S −5.7 GS ALEEKKGNYVVTDHGSCV 5.345 S−5.7 IAMGFPQKDLKAYTGTIL 9.625 S −4 AAVDSVTIPPAQCYLSLLHL 8.895 S −5.9QQRRMQSA PAAVDSVTIPPAQCYLSLLHL 4.935 S −5.9 DLSYVSDQNGGVPDQILLHL 3.765 S−7.7 RPTED AVRSPGSPLILEVGSGSGAIS 6.975 S −5.4 LEEVAQRSHAVRSPGSPLILE5.395 S −5.4 VG LAALCPASRALEEKKGNYVV 7.155 S −5.7 TDHGSLAALCPASRALEEKKGNYVV 7.155 S −5.7 TDH ASRALEEKKGNYVVTDHGS 8.845 S −5.7CVRA ALCPASRALEEKKGNYVV 8.845 S −5.3 AALCPASRALEEKKGNYV 8.845 S −3.8SHHTHSYQRYSHPLFLPGHR 9.585 S −6.1 LDPPI SHQIHSYQLYTHPLLHPWD 6.605 S −5HRD DKGHQFHVHPLLHSGDDLD 5.565 S −5 P KLRTIPLSDNTIFRRICTIAKH 10.565 S−5.5 LE ASATEPANDSLFSPGAANLF 4.075 S −5 STYLAR FPVVQSTEDVFPQGLPNEY 2.945S −7.2 AFVT AASAAAFPSQRTSWEFLQSL 9.885 S −4.3 VSIKQEKGSVLQFMPFTTVSELMKVS 9.885 S −4.8 AMSSPKV NQVLASRYGIRGFSTIKIFQK 10.695 S−4.3 GESPV ARLQSKEYPVIFKSIMRQRLI 11.405 S −5.8 SPQLDVTGPHLYSIYLHGSTDKLPY 6.015 S −6.4 VTMGS SHLASLKNNVSPVLRSHSFS 10.585 S−3.3 DPSPKFA TAQFAPSPGQPPALSPSYPG 9.845 S −3 HRLPLQQGpASAKSRREFDKIELAYRR 10.675 S −4.6 MAGPKGFQYRALYPFRRER 11.265 S −4.6SDAFSGLTALPQSILLFGP 3.095 S −7.9 STQHADLTIIDNIKEMNFLR 9.625 S −5.8 RYKLHTHYDYVSALHPVSTPSKE 6.305 S −5.5 YTSA SSPLGRANGRRFANPRDSFS 12.575 S −3AMGFQR EIHGKCENMTITSRGTTVTP 7.165 S −3.9 TKETVSLG LNTGLFRIKFKEPLENLI9.885 S −4.3 SPQSGGAATLAAQARLQPV 6.035 S −4.9 HLDVWGEHERGGSGSQMPAWRTRGAISASS 12.705 S −3.9 TQKTPTTRL GLTRISIQRAQPLPPCLPSFR 12.105S −2.8 PPTALQGLS SRLQTRKNKKLALSSTPSNIA 11.565 S −4.1 PSDWCTEMKRVFGFPVHYTDVS 7.155 S −4.8 NMS GPLQLPVTRKNMPLPGVVK 11.635 S −3LPPLPGS ALLQNVELRRNVLVSPTPLA 10.885 S −4.8 N VNGISSQPQVPFYPNLQKS 9.395 S−4.8 QYYSTV YLSHTLGAASSFMRPTVPPP 9.845 S −4.1 QF SLRNNMFEISDRFIGIYKTYN9.935 S −4.3 ITK VTLNDMKARQKALVRERER 11.305 S −3.8 QLAVKQLERGEASVVDFKKNLEY 7.095 S −3.7 AAT TKLKSKAPHWTNCILHEYKN 9.965 S −5.1LSTS FAKGFRESDLNSWPVAPRP 10.085 S −3.6 LLSV HLLQKQTSIQSPSLYGNSSPP 10.175S −4.1 LNK STEVEPKESPHLARHRHLMK 10.315 S −3.7 TLVKSLSTDGAWPVLLDKFVEWYKDK 4.455 S −5.7 QMS SHKLESIKEITNFKDAKQLL 9.665 S −3.6TGKPEMDFVRLAQLFARAR 11.225 S −4.8 PMGLF

FIG. 15 plots those parameters for this set of peptides on the x (P_(i))and y (HYDRO) axes. As observed, insoluble peptides are distributedthroughout the x-y space while soluble peptides are observed in morediscrete regions. Thus, solubility is determined by a balance of netcharge and hydrophobicity and can be predictable based on the amino acidsequence.

The % of peptides that are soluble differs by region. In FIG. 15, regionA is bounded by Pi ≥5 and HYDRO ≥−6.0 and Pi ≥8 and HYDRO ≥−8.0, regionB is bounded by Pi ≤5 and HYDRO ≥−5, and region C is bounded by Pi ≥9and HYDRO ≤−8.0. In the preferred regions (A, B and C), the % ofpeptides examined that were soluble are specified in Table 13 and rangefrom 64% to 89%. In the non-preferred regions (“Other”), only about42.5% of the peptides were soluble.

TABLE 13 A B C Other # Soluble 115 25 9 17 # Insoluble 15 3 4 23 %Soluble 88% 89% 64% 42.5%

An Excel spreadsheet can be built that allows alterations to the lengthor specific sequence of a peptide region and immediate re-calculation ofthese values for the selected peptide. This approach can facilitatedesign of a peptide with higher predicted solubility or rejection ofpotential peptides as unlikely to be soluble. Such an approach cansignificantly benefit peptide manufacturers who desire to producesoluble peptides.

This approach was developed with a particular aqueous formulation (D5W/5mM succinate) but can readily be adapted to any other aqueousformulation to identify the appropriate combination of P_(i) andhydrophobicity.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

1. A pharmaceutical composition comprising: (a) at least oneneo-antigenic peptide or a pharmaceutically acceptable salt thereof; (b)a pH modifier; and (c) a pharmaceutically acceptable carrier; whereinthe at least one neo-antigenic peptide or pharmaceutically acceptablesalt thereof is bounded by Pi ≥5 and HYDRO ≥−6.0, Pi ≥8 and HYDRO ≥−8.0,Pi ≤5 and HYDRO ≥−5, and Pi ≥9 and HYDRO ≤−8.0, or Pi >7 and a HYDROvalue of ≥−5.5.
 2. The pharmaceutical composition of claim 1, whereinthe pharmaceutical composition is a vaccine composition.
 3. Thepharmaceutical composition of claim 1, wherein the at least oneneo-antigenic peptide or the pharmaceutically acceptable salt thereof isbound by Pi >7 and a HYDRO value of ≥−5.5.
 4. The pharmaceuticalcomposition claim 1, wherein the pharmaceutical composition comprises atleast two, three, four, or five neo-antigenic peptides. 5-6. (canceled)7. The pharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition comprises up to 40 neo-antigenic peptides. 8-9. (canceled)10. The pharmaceutical composition of claim 1, wherein the at least oneneoantigenic peptide ranges from 5 to 50 amino acids in length, 15 to 35amino acids in length, 15 to 24 amino acids in length, 6 to 25 aminoacids in length, 9 to 15 amino acids in length, 8 to 11 amino acids inlength, or 9 or 10 amino acids in length. 11-12. (canceled)
 13. Thepharmaceutical composition of claim 1, wherein the pH modifier is abase.
 14. The pharmaceutical composition of claim 1, wherein the pHmodifier is a dicarboxylate or tricarboxylate salt.
 15. Thepharmaceutical composition of claim 1, wherein the pH modifier issuccinate or citrate.
 16. (canceled)
 17. The pharmaceutical compositionof claim 1, wherein the pH modifier is sodium succinate.
 18. Thepharmaceutical composition of claim 15, wherein succinate is present inthe formulation at a concentration from about 1 mM to about 10 mM. 19.The pharmaceutical composition of claim 18, wherein succinate is presentin the formulation at a concentration of about 2 mM to about 5 mM. 20.The pharmaceutical composition of claim 1, wherein the pharmaceuticallyacceptable carrier comprises water.
 21. The pharmaceutical compositionof claim 1, wherein the pharmaceutically acceptable carrier furthercomprises dextrose, trehalose, or sucrose. 22-23. (canceled)
 24. Thepharmaceutical composition of claim 1, wherein the pharmaceuticallyacceptable carrier further comprises dimethylsulfoxide.
 25. Thepharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition is lyophilizable.
 26. The pharmaceutical composition ofclaim 1, wherein the pharmaceutical composition further comprises animmunomodulator or adjuvant.
 27. The pharmaceutical composition of claim26, wherein the immunodulator or adjuvant is selected from the groupconsisting of poly-ICLC, 1018 ISS, aluminum salts, Amplivax, AS15, BCG,CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFactIMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59,monophosphoryllipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®,vector system, PLGA microparticles, resiquimod, SRL172, Virosomes andother Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan,Pam3Cys, and Aquila's QS21 stimulon.
 28. The pharmaceutical compositionof claim 26, wherein the immunomodulator or adjuvant comprisespoly-ICLC. 29-30. (canceled)
 31. A method of preparing a neo-antigenicpeptide solution for a neoplasia vaccine, the method comprising: (a)preparing a solution comprising at least one neo-antigenic peptide or apharmaceutically acceptable salt thereof, wherein the at least oneneo-antigenic peptide or pharmaceutically acceptable salt thereof isbounded by Pi ≥5 and HYDRO ≥−6.0, Pi ≥8 and HYDRO ≥−8.0, Pi ≤5 and HYDRO≥−5, and Pi ≥9 and HYDRO ≤−8.0, or Pi >7 and a HYDRO value of ≥−5.5; and(b) combining the solution comprising at least one neo-antigenic peptideor a pharmaceutically acceptable salt thereof with a solution comprisingsuccinic acid or a pharmaceutically acceptable salt thereof, therebypreparing a peptide solution for a neoplasia vaccine. 32-38. (canceled)39. A method of treating a subject diagnosed as having a neoplasia, themethod comprising administering the pharmaceutical composition of claim1 to the subject, thereby treating the neoplasia.
 40. The method ofclaim 39, further comprising administering a second, third, or fourthpharmaceutical composition of claim 1 to the subject. 41-44. (canceled)45. A vaccination or immunization kit comprising: (a) a separatelypackaged freeze-dried immunogenic composition configured to elicit animmune response to at least one neoantigen; and (b) a solution for thereconstitution of the freeze-dried vaccine, wherein the immunogeniccomposition comprises at least one neo-antigenic peptide orpharmaceutically acceptable salt thereof bounded by Pi ≥5 and HYDRO≥−6.0, Pi ≥8 and HYDRO ≥−8.0, Pi ≤5 and HYDRO ≥−5, and Pi ≥9 and HYDRO≤−8.0, or Pi >7 and a HYDRO value of ≥−5.5. 46-103. (canceled)